Epidemiology and molecular characterization of Mycobacterium tuberculosis including a drug-resistant strain associated with mortality of Asian elephants in Nepal 2019-2022.

Tuberculosis (TB) is an emerging threat to the survival of elephants in Nepal. We investigated the lung tissue samples from nine elephants that died from 2019 to 2022 in Nepal using culture, conventional PCR, and loop-mediated isothermal amplification (LAMP) and then performed genotyping of five PCR-positive isolates to understand the possible transmission dynamics of Mycobacterium tuberculosis (Mtb). Results showed that two-thirds (6/9) of elephants were confirmed to be infected from Mtb by LAMP, 5/9 by PCR, and 4/9 by culture. Genotyping of Mtb isolates showed that elephants were infected with the Indo-Oceanic and Beijing lineages including an isoniazid-resistant Beijing lineage. MIRU-VNTR-based phylogeny, gyrA, and katG sequencing showed the possibility of ongoing transmission of Indo-Oceanic lineages and likely transmission of the drug-resistant Beijing lineage from human to elephant. Implementation of comprehensive surveillance and preventive measures are urgently needed to address this zoonotic disease and protect elephants from TB in Nepal.

Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS.

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml-1, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.

Four-Month High-Dose Rifampicin Regimens for Pulmonary Tuberculosis.

BACKGROUND: Shorter but effective tuberculosis treatment regimens would be of value to the tuberculosis treatment community. High-dose rifampicin has been associated with more rapid and secure lung sterilization and may enable shorter tuberculosis treatment regimens. METHODS: We randomly assigned adults who were given a diagnosis of rifampicin-susceptible pulmonary tuberculosis to a 6-month control regimen, a similar 4-month regimen of rifampicin at 1200 mg/d (study regimen 1 [SR1]), or a 4-month regimen of rifampicin at 1800 mg/d (study regimen 2 [SR2]). Sputum specimens were collected at regular intervals. The primary end point was a composite of treatment failure and relapse in participants who were sputum smear positive at baseline. The noninferiority margin was 8 percentage points. Using a sequence of ordered hypotheses, noninferiority of SR2 was tested first. RESULTS: Between January 2017 and December 2020, 672 patients were enrolled in six countries, including 191 in the control group, 192 in the SR1 group, and 195 in the SR2 group. Noninferiority was not shown. Favorable responses rates were 93, 90, and 87% in the control, SR1, and SR2 groups, respectively, for a country-adjusted absolute risk difference of 6.3 percentage points (90% confidence interval, 1.1 to 11.5) comparing SR2 with the control group. The proportions of participants experiencing a grade 3 or 4 adverse event were 4.0, 4.5, and 4.4% in the control, SR1, and SR2 groups, respectively. CONCLUSIONS: Four-month high-dose rifampicin regimens did not have dose-limiting toxicities or side effects but failed to meet noninferiority criteria compared with the standard 6-month control regimen for treatment of pulmonary tuberculosis. (Funded by the MRC/Wellcome Trust/DFID Joint Global Health Trials Scheme; ClinicalTrials.gov number, NCT02581527.)

Multidrug-resistant and extended-spectrum beta-lactamase-producing uropathogens in children in Bhaktapur, Nepal.

BACKGROUND: The emergence of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing uropathogens has complicated the treatment of urinary tract infections (UTI). Paediatric UTI is a common illness, which if not treated properly, may lead to acute and long-term complications, such as renal abscess, septicaemia, and renal scarring. This study aimed to determine the prevalence of MDR and ESBL-producing uropathogens among children. METHODS: During the study period (April 2017-April 2018), midstream urine samples were collected following aseptic procedures from children < 16 years in Siddhi Memorial Hospital. Standard culture and biochemical tests were performed to identify uropathogens and antimicrobial susceptibility test was done by modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL-producing uropathogens were screened by ceftazidime (30 µg) and cefotaxime (30 µg) discs, and confirmed by the combination disc tests: ceftazidime + clavulanic acid (30/10 µg) or cefotaxime + clavulanic acid (30/10 µg) as recommended by CLSI. RESULTS: We processed 5545 non-repeated urine samples from the children with symptoms of UTI. A significant growth of uropathogens was observed in 203 samples (3.7%). The median age of the children was 24 months (interquartile range (IQR), 12-53 months). Escherichia coli (n = 158, 77.8%) and Klebsiella pneumoniae (n = 30, 14.8%) were common among the uropathogens. Among them, 80.3% were resistant to amoxycillin and 51.2% were resistant to cotrimoxazole. Most of them were susceptible to amikacin, nitrofurantoin, and ofloxacin. MDR was detected in 34.5% (n = 70/203) and ESBL producers in 24.6% (n = 50/203) of them. The proportion of MDR isolates was higher in children < 5 years (n = 59/153, 38.6%) than children ≥ 5 years (n = 11/50, 22%) (P = 0.03). CONCLUSIONS: Nitrofurantoin, ofloxacin, and amikacin can be used for the empirical treatment for UTI in children in Bhaktapur, Nepal. MDR and ESBL-producing uropathogens are prevalent; this warrants a continuous surveillance of antimicrobial resistance.