Direct Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis by Use of pncA PCR Sequencing.

An in-house-developed pncA sequencing assay for analysis of pyrazinamide (PZA) resistance was evaluated using 162 archived Mycobacterium tuberculosis complex (MTBC) isolates with phenotypic PZA susceptibility profiles that were well defined by analysis of Bactec MGIT 960 PZA kit and PZase activity data. Preliminary results showed 100% concordance between pncA sequencing and phenotypic PZA drug susceptibility test (DST) results among archived isolates. Also, 637 respiratory specimens were prospectively collected, and 158 were reported as MTBC positive by the Abbott Realtime MTB assay (96.3% sensitivity [95% confidence interval {CI}: 92.2% to 98.7%]; 100% specificity [95% CI: 99.2% to 100.0%]). Genotypic and phenotypic PZA resistance profiles of these 158 MTBC-positive specimens were analyzed by pncA sequencing and Bactec MGIT 960 PZA kit, respectively. For analysis of PZA resistance, pncA sequencing detected pncA mutations in 5/5 (100%) phenotypic PZA-resistant respiratory specimens within 4 working days. No pncA mutations were detected among PZA-susceptible specimens. Combining archived isolates with prospective specimens, 27 were identified as phenotypic PZA resistant with pncA mutation. Among these 27 samples, 6/27 (22.2%) phenotypic PZA-resistant strains carried novel pncA mutations without rpsA and panD mutations. These included 5 with mutations (a deletion [Del] at 383T [Del383T], Del 380 to 390, insertion of A [A Ins] at position 127, A Ins at position 407, and G Ins at position 508) in pncA structural genes and 1 with a mutation (T-12C) at the pncA promoter region. All six of these strains had no or reduced PZase activities, indicating that the novel mutations might confer PZA resistance. Additionally, 25/27 phenotypic PZA-resistant strains were confirmed multidrug-resistant tuberculosis (MDR-TB) strains. As PZA is commonly used in MDR-TB treatment regimens, direct pncA sequencing will rapidly detect PZA resistance and facilitate judicious use of PZA in treating PZA-susceptible MDR-TB.

Zika virus infection-the next wave after dengue?

Zika virus was initially discovered in east Africa about 70 years ago and remained a neglected arboviral disease in Africa and Southeast Asia. The virus first came into the limelight in 2007 when it caused an outbreak in Micronesia. In the ensuing decade, it spread widely in other Pacific islands, after which its incursion into Brazil in 2015 led to a widespread epidemic in Latin America. In most infected patients the disease is relatively benign. Serious complications include Guillain-Barré syndrome and congenital infection which may lead to microcephaly and maculopathy. Aedes mosquitoes are the main vectors, in particular, Ae. aegypti. Ae. albopictus is another potential vector. Since the competent mosquito vectors are highly prevalent in most tropical and subtropical countries, introduction of the virus to these areas could readily result in endemic transmission of the disease. The priorities of control include reinforcing education of travellers to and residents of endemic areas, preventing further local transmission by vectors, and an integrated vector management programme. The container habitats of Ae. aegypti and Ae. albopictus means engagement of the community and citizens is of utmost importance to the success of vector control.

Ebola virus disease in nonendemic countries.

The 2014 West African outbreak of Ebola virus disease was unprecedented in its scale and has resulted in transmissions outside endemic countries. Clinicians in nonendemic countries will most likely face the disease in returning travelers, either among healthcare workers, expatriates, or visiting friends and relatives. Clinical suspicion for the disease must be heightened for travelers or contacts presenting with compatible clinical syndromes, and strict infection control measures must be promptly implemented to minimize the risk of secondary transmission within healthcare settings or in the community. We present a concise review on human filoviral disease with an emphasis on issues that are pertinent to clinicians practicing in nonendemic countries.

Mycolyltransferase is important for biofilm formation and pathogenesis of Tsukamurella keratitis.

Tsukamurella, a group of multi-drug resistant, Gram-positive, aerobic, and partially acid-fast bacteria, are emerging causes of bacterial conjunctivitis and keratitis. However, the pathogenesis of Tsukamurella keratitis is largely unknown. To address this, we used New Zealand White rabbits to develop the first eye infection model and conducted in vitro tests to study the pathogenesis mechanisms of Tsukamurella. There is increasing evidence that biofilms play a significant role in ocular infections, leading us to hypothesize that biofilm formation is crucial for effective Tsukamurella infection. In order to look for potential candidate genes which are important in biofilm formation and Tsukamurella keratitis. We performed genome sequencing of two ocular isolates, T. pulmonis-PW1004 and T. tyrosinosolvens-PW899, to identify potential virulence factors. Through in vitro and in vivo studies, we characterized their biological roles in mediating Tsukamurella keratitis. Our findings confirmed that Tsukamurella is an ocular pathogen by fulfilling Koch's postulates, and using genome sequence data, we identified tmytC, encoding a mycolyltransferase, as a crucial gene in biofilm formation and causing Tsukamurella keratitis in the rabbit model. This is the first report demonstrating the novel role of mycolyltransferase in causing ocular infections. Overall, our findings contribute to a better understanding of Tsukamurella pathogenesis and provide a potential target for treatment. Specific inhibitors targeting TmytC could serve as an effective treatment option for Tsukamurella infections.

Genetic mechanisms of co-emergence of INH-resistant Mycobacterium tuberculosis strains during the standard course of antituberculosis therapy.

The incidence of isoniazid (INH) resistant Mycobacterium tuberculosis is increasing globally. This study aimed to identify the molecular mechanisms behind the development of INH resistance in M. tuberculosis strains collected from the same patients during the standard course of treatment. Three M. tuberculosis strains were collected from a patient before and during antituberculosis (anti-TB) therapy. The strains were characterized using phenotypic drug susceptibility tests, Mycobacterial Interspersed Repeated Unit-Variable-Number Tandem Repeats (MIRU-VNTR), and whole-genome sequencing (WGS) to identify mutations associated with INH resistance. To validate the role of the novel mutations in INH resistance, the mutated katG genes were electroporated into a KatG-deleted M. tuberculosis strain (GA03). Three-dimensional structures of mutated KatG were modeled to predict their impact on INH binding. The pre-treatment strain was susceptible to INH. However, two INH-resistant strains were isolated from the patient after anti-TB therapy. MIRU-VNTR and WGS revealed that the three strains were clonally identical. A missense mutation (P232L) and a nonsense mutation (Q461Stop) were identified in the katG of the two post-treatment strains, respectively. Transformation experiments showed that katG of the pre-treatment strain restored INH susceptibility in GA03, whereas the mutated katG genes from the post-treatment strains rendered negative catalase activity and INH resistance. The protein model indicated that P232L reduced INH-KatG binding affinity while Q461Stop truncated gene transcription. Our results showed that the two katG mutations, P232L and Q461Stop, accounted for the co-emergence of INH-resistant clones during anti-TB therapy. The inclusion of these mutations in the design of molecular assays could increase the diagnostic performance.IMPORTANCEThe evolution of drug-resistant strains of Mycobacterium tuberculosis within the lung lesions of a patient has a detrimental impact on treatment outcomes. This is particularly concerning for isoniazid (INH), which is the most potent first-line antimycobacterial drug. However, the precise genetic factors responsible for drug resistance in patients have not been fully elucidated, with approximately 15% of INH-resistant strains harboring unknown genetic factors. This raises concerns about the emergence of drug-resistant clones within patients, further contributing to the global epidemic of resistance. In this study, we revealed the presence of two novel katG mutations, which emerged independently due to the stress exerted by antituberculosis (anti-TB) treatment on a parental strain. Importantly, we experimentally demonstrated the functional significance of both mutations in conferring resistance to INH. Overall, this research sheds light on the genetic mechanisms underlying the evolution of INH resistance within patients and provides valuable insights for improving diagnostic performance by targeting specific mutations.

Bacillus bloodstream infections in a tertiary perinatal centre: an 8-year study.

BACKGROUND: Isolates of nonanthrax Bacillus species in clinical samples are frequently considered as contaminants. However, there were case reports describing Bacillus sepsis among infants, associated with high mortality and morbidity. METHODS: We performed a retrospective review of the clinical and epidemiological features of Bacillus bacteremia at our neonatal intensive care unit from January 2002 to December 2009. RESULTS: Bacillus bacteremia was considered to be clinically significant in 11 infants. The median gestational age was 30 weeks. All had either central catheters or peripherally inserted arterial lines in situ. The mean neutrophil and lymphocyte counts were 6.73 × 10(9)/L (0.78 to 12.56 × 10(9)/L) and 2.75 × 10(9)/L (0.82 to 6.15 × 10(9)/L), respectively. All 11 infants received intravenous vancomycin, with an average duration of 12.4 days. In general, the earlier the catheter was removed, the quicker the clearance of bacteremia was achieved. All infants survived and were discharged from the hospital. CONCLUSIONS: The growth of Bacillus species in blood cultures cannot simply be regarded as a contaminant. Hematologic parameters are frequently unremarkable at the disease onset. Increased vigilance, early diagnosis, and effective therapy in conjunction with prompt catheter removal are the keys to successful management of Bacillus bacteremia.

Infections associated with body modification.

Although exact statistics are lacking, body modifications for cosmetic purposes are performed in many countries. The commonest forms include tattooing, body piercing, and breast and facial augmentation using implants or injectable fillers. Liposuction and, to a lesser extent, mesotherapy are also practiced in many countries. Infective complications of these procedures include local infections, transmission of bloodborne pathogens (viral hepatitis and human immunodeficiency virus), and distant infections such as infective endocarditis. Presence of foreign bodies, long healing time of piercing wounds, and poor compliance with infection control practices of some practitioners all predispose the recipients to infections. Apart from the endogenous microbial flora of the skin and mucosae, atypical mycobacteria, especially the rapid growers, have emerged as some of the most important pathogens in such settings. Outbreaks of infection are commonly reported. We hereby review the current knowledge of the topic with specific focus on infections associated with tattooing, body piercing, breast augmentation, mesotherapy, liposuction, and tissue filler injections. Greater awareness among consumers and health-care professionals, as well as more stringent regulations by the health authorities, is essential to minimize the health risks arising from these procedures.

Risk Factors for Slow Viral Decline in COVID-19 Patients during the 2022 Omicron Wave.

Formulating termination of isolation (de-isolation) policies requires up-to-date knowledge about viral shedding dynamics. However, current de-isolation policies are largely based on viral load data obtained before the emergence of Omicron variant. In this retrospective cohort study involving adult patients hospitalised for COVID-19 between January and February 2022, we sought to determine SARS-CoV-2 viral shedding kinetics and to investigate the risk factors associated with slow viral decline during the 2022 Omicron wave. A total of 104 patients were included. The viral load was highest (Ct value was lowest) on days 1 post-symptom-onset (PSO) and gradually declined. Older age, hypertension, hyperlipidaemia and chronic kidney disease were associated with slow viral decline in the univariate analysis on both day 7 and day 10 PSO, while incomplete or no vaccination was associated with slow viral decline on day 7 PSO only. However, older age was the only risk factor that remained statistically significant in the multivariate analysis. In conclusion, older age is an independent risk factor associated with slow viral decline in this study conducted during the Omicron-dominant 2022 COVID-19 wave. Transmission-based precaution guidelines should take age into consideration when determining the timing of de-isolation.

Diagnostic evaluation of an in-house developed single-tube, duplex, nested IS6110 real-time PCR assay for rapid pulmonary tuberculosis diagnosis.

OBJECTIVE: To perform a prospective evaluation on the diagnostic performance of an in-house developed, duplex nested IS6110 real-time Polymerase-Chain-Reaction (PCR) assay (IS6110-qPCR assay) for rapid pulmonary TB diagnosis. METHODS: A total of 503 sputum specimens were prospectively collected from July 2016 to November 2016. Diagnostic accuracy and optimal cut-off Cycle-threshold (Ct) value for IS6110-qPCR assay was determined by Receiver Operating Characteristic (ROC) curve. Using the optimal cut-off Ct, diagnostic performance of IS6110-qPCR assay was assessed with reference to both bacteriological and clinical information. Meanwhile, limit of detection (LOD) was calculated using Mycobacterium tuberculosis H37Rv as reference strain. RESULT: ROC curve analysis of IS6110-qPCR assay showed a high Area Under the Curve (AUC) value (0.948) with optimal Ct value at 24.140. Prospective analysis of IS6110-qPCR assay with cut-off Ct = 24.140 showed a high overall sensitivity and specificity of 97.2% and 99.7%, respectively. No cross reactivity was observed among all non-tuberculous mycobacteria specimens in this study. LOD analysis on MTB-spiked sputum showed an average detection limit of 5.0 CFU/mL at Ct = 23.18 (±SD, 0.57). CONCLUSION: IS6110-qPCR assay is a highly accurate and cost-effective assay developed for primary screening of suspected TB cases, which is particularly suitable for regions with limited resources but high TB burden.

Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c (lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c (cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA, and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

Direct detection of Mycobacterium tuberculosis and drug resistance in respiratory specimen using Abbott Realtime MTB detection and RIF/INH resistance assay.

Abbott RealTime MTB (Abbott-RT) in conjunction with Abbott RealTime MTB RIF/INH Resistance (Abbott-RIF/INH) is a new, high-throughput automated nucleic acid amplification platform (Abbott-MDR) for detection of Mycobacterium tuberculosis complex (MTBC) and the genotypic markers for rifampicin (RIF) and isoniazid (INH) resistance directly from respiratory specimens. This prospective study evaluated the diagnostic performance of this new platform for MTBC and multidrug-resistant tuberculosis (MDR-TB) using 610 sputum specimens in a tuberculosis high-burden setting. Using conventional culture results and clinical background as reference standards, Abbott-RT exhibited an overall sensitivity and specificity of 95.2% and 99.8%, respectively. Genotypic RIF/INH resistance of 178 "MTB detected" specimens was subsequently analyzed by Abbott-RIF/INH. Compared to phenotypic drug susceptibility test results, Abbott-RIF/INH detected resistance genotypic markers in 84.6% MDR-TB, 80% mono-RIF-resistant and 66.7% mono-INH-resistant specimens. Two of the RIF-resistant specimens carried a novel single, nonsense mutation at rpoB Q513 and in silico simulation demonstrated that the truncated RpoB protein failed to bind with other subunits for transcription. Overall, Abbott-MDR platform provided high throughput and reliable diagnosis of MDR-TB within a TB high-burden region.

An unprecedented outbreak investigation for nosocomial and community-acquired legionellosis in Hong Kong.

BACKGROUND: The environmental sources associated with community-acquired or nosocomial legionellosis were not always detectable in the mainland of China and Hong Kong, China. The objective of this study was to illustrate the control measures implemented for nosocomial and community outbreaks of legionellosis, and to understand the environmental distribution of legionella in the water system in Hong Kong, China. METHODS: We investigated the environmental sources of two cases of legionellosis acquired in the hospital and the community by extensive outbreak investigation and sampling of the potable water system using culture and genetic testing at the respective premises. RESULTS: The diagnosis of nosocomial legionellosis was suspected in a patient presenting with nosocomial pneumonia not responsive to multiple beta-lactam antibiotics with subsequent confirmation by Legionella pneumophila serogroup 1 antigenuria. High counts of Legionella pneumophila were detected in the potable water supply of the 70-year-old hospital building. Another patient on continuous ambulatory peritoneal dialysis presenting with acute community-acquired pneumonia and severe diarrhoea was positive for Legionella pneumophila serogroup 1 by polymerase chain reaction (PCR) testing on both sputum and nasopharyngeal aspirate despite negative antigenuria. Paradoxically the source of the second case was traced to the water system of a newly commissioned office building complex. No further cases were detected after shock hyperchlorination with or without superheating of the water systems. Subsequent legionella counts were drastically reduced. Point-of-care infection control by off-boiled or sterile water for mouth care and installation of water filter for showers in the hospital wards for immunocompromised patients was instituted. Territory wide investigation of the community potable water supply showed that 22.1% of the household water supply was positive at a mean legionella count of 108.56 CFU/ml (range 0.10 to 639.30 CFU/ml). CONCLUSIONS: Potable water systems are open systems which are inevitably colonized by bacterial biofilms containing Legionella species. High bacterial counts related to human cases may occur with stagnation of flow in both old or newly commissioned buildings. Vigilance against legionellosis is important in healthcare settings with dense population of highly susceptible hosts.

Review article: Mycobacterium marinum infection of the hand and wrist.

Misdiagnosis and delayed treatment of Mycobacterium marinum infection is common because of its diverse manifestations. This leads to inappropriate use of antimicrobials, extension of the infection from the skin to the tenosynovium, and a poor prognosis (loss of tendons and prolonged immobilisation, secondary to multiple debridements and joint contractures). Clinicians should be aware of this type of infection, especially in subjects at risk (fishermen and aquarium enthusiasts), and those with a history of trauma coupled with exposure to water or marine life. A proactive approach to obtain a biopsy for histopathological and microbiological diagnosis is advised. Anti-mycobacterial treatment should be started promptly. The combined use of rifampicin, ethambutol, and clarithromycin appears to be effective, and debridement is indicated in patients with deep-seated infections.

Diagnosis and spectrum of melamine-related renal disease: plausible mechanism of stone formation in humans.

BACKGROUND: An epidemic of urinary stones affecting children after consumption of melamine tainted milk is unfolding. We defined clinicopathological features of the disease for diagnosis, monitoring, and treatment of this group of patients. METHODS: A clinicopathological study on exposed children with ultrasonographic evidence of urolithiasis was conducted. Melamine and cyanuric acid levels in the urine were determined by mass spectrometry. RESULTS: Disease severity varied from acute renal failure with hydronephrosis to symptomatic or asymptomatic stones with or without abnormal urinalysis. All cases were aged <3 y with >50% cases having predisposing urinary metabolic risk factors for urolithiasis. Most of the stones were located in the renal pelvis and measured 2.5-18 mm by ultrasonography. We found a strong correlation between renal stone size and urinary melamine concentration. For stones <10 mm, a 10 microg/mmol creatinine increase in urinary melamine concentration is associated with approximately 1 mm increase in the size of the stone. The high degree of correlation strongly suggests that melamine is related to stone formation in humans. Using ROC analysis, we propose that patients who have a persistent melamine level above the optimal cut-off value of 7.1 microg melamine/mmol creatinine in urine might have a significant exposure of melamine-tainted products. Unlike melamine, urinary cyanuric acid is not significantly different between cases and controls. Pathophysiological findings from feeding animals with melamine and cyanuric acid may not be directly applicable to humans. CONCLUSION: Both melamine and urine metabolic lithogenic factors are important for the formation of melamine-related stones. Apart from aiding with case screening and confirmation, the urine melamine level might as well be an indicator of residual melamine load in the body and thus is useful for following-up and monitoring of the confirmed cases. As the stones are small and can be passed out spontaneously, follow-up of these patients with urine melamine will be a convenient tool for monitoring the melamine load of the patients.

CTX-M type beta-lactamases among fecal Escherichia coli and Klebsiella pneumoniae isolates in non-hospitalized children and adults.

We investigated the occurrence and diversity of extended-spectrum beta-lactamase (ESBL) enzymes among antibiotic-resistant Escherichia coli and Klebsiella pneumoniae isolates obtained from human feces. All ESBL-positive isolates were characterized at the molecular level by polymerase chain reaction, sequencing and pulsed-field gel electrophoresis (PFGE). Eight of 46 antibiotic-resistant E. coli (6 from children and 2 from adults) and 4 of 8 K. pneumoniae (all from adults) isolates were found to be ESBL-positive by the double-disk synergy test. Seven isolates were found to have CTX-M-14, 2 each had CTX-M-24 and CTX-M-38, and 1 had CTX-M-9. In addition, 8 isolates were found to carry TEM-1b or TEM-1c. No SHV-type enzyme was found among the E. coli strains. In 9 strains, the plasmidic bla(CTX-M) determinants were transferable to E. coli by conjugation. Analysis by PFGE showed evidence of clonal and non-clonal spread. The present study shows fecal carriage of organisms producing bla(CTX-M) determinants and underscores the role that commensals could play as a reservoir for their dissemination.