Xpert MTB/RIF Ultra Is Highly Sensitive for the Diagnosis of Tuberculosis Lymphadenitis in a High-HIV Setting.

Tuberculosis lymphadenitis (TBL) is the most common extrapulmonary tuberculosis (EPTB) manifestation. Xpert MTB/RIF Ultra (Ultra) is a World Health Organization-endorsed diagnostic test, but performance data for TBL, including on noninvasive specimens, are limited. Fine-needle aspiration biopsy specimens (FNABs) from outpatients (≥18 years) with presumptive TBL (n = 135) underwent (i) routine Xpert MTB/RIF testing (later with Ultra once programmatically available), (ii) MGIT 960 culture (if Xpert or Ultra negative or rifampicin resistant), and (iii) study Ultra testing. Concentrated paired urine specimens underwent Ultra testing. Primary analyses used a microbiological reference standard (MRS). In a head-to-head comparison (n = 92) of an FNAB study Ultra and Xpert, Ultra had increased sensitivity (91% [95% confidence interval: 79, 98] versus 72% [57, 84]; P = 0.016) and decreased specificity (76% [61, 87] versus 93% [82, 99]; P = 0.020) and diagnosed patients not on treatment. Neither HIV nor alternative reference standards affected sensitivity and specificity. In patients with both routine and study Ultra tests, the latter detected more cases (+20% [0, 42]; P = 0.034), and false-negative study Ultra results were more inhibited than true-positive results. Study Ultra false positives had less mycobacterial DNA than true positives (trace-positive proportions, 59% [13/22] versus 12% [5/51]; P < 0.001). "Trace" exclusion or recategorization removed potential benefits offered over Xpert. Urine Ultra tests had low sensitivity (18% [7, 35]). Ultra testing on FNABs is highly sensitive and detects more TBL than Xpert (Ultra still missed some cases due in part to inhibition). Patients with FNAB Ultra-positive "trace" results, most of whom will be culture negative, may require additional clinical investigation. Urine Ultra testing could reduce the number of patients needing invasive sampling.

Genetic and Morphological Characterization of Freshwater Shrimps (Caridina africana Kingsley, 1882) Reveals the Presence of Alien Shrimps in the Cape Floristic Region, South Africa.

Morphological identification and molecular data (mtDNA COI) were used to resolve the taxonomic identity of a non-native freshwater shrimp in the Cape Floristic Region (CFR) of South Africa and to evaluate levels of genetic diversity and differentiation in the species' core natural distribution. The species was morphologically and genetically identified as Caridina africana Kingsley, 1882, whose main natural distribution is in the KwaZulu-Natal (KZN) Province, more than 1200 km from the point of new discovery. Subsequently, sequence data from natural populations occurring in seven rivers throughout KZN showed the presence of nuclear copies of the mtDNA COI gene (NUMTs) in 46 out of 140 individuals. Upon removal of sequences containing NUMTs, levels of genetic diversity were low in the alien population (possibly as a consequence of a bottleneck event), while varying levels of genetic diversity and differentiation were found in natural populations, indicating habitat heterogeneity, fragmentation and restricted gene flow between rivers. Following the present study, the alien shrimp has survived the Western Cape's winter and dispersed into a nearby tributary of the Eerste River System, hence posing an additional potential threat to endangered endemics. Understanding the biology of this alien species will aid detection and eradication procedures.

Subtracting the background by reducing cell-free DNA's confounding effects on Mycobacterium tuberculosis quantitation and the sputum microbiome.

DNA characterisation in people with tuberculosis (TB) is critical for diagnostic and microbiome evaluations. However, extracellular DNA, more frequent in people on chemotherapy, confounds results. We evaluated whether nucleic acid dyes [propidium monoazide (PMA), PEMAX] and DNaseI could reduce this. PCR [16S Mycobacterium tuberculosis complex (Mtb) qPCR, Xpert MTB/RIF] was done on dilution series of untreated and treated (PMA, PEMAX, DNaseI) Mtb. Separately, 16S rRNA gene qPCR and sequencing were done on untreated and treated sputa before (Cohort A: 11 TB-negatives, 9 TB-positives; Cohort B: 19 TB-positives, PEMAX only) and 24-weeks after chemotherapy (Cohort B). PMA and PEMAX reduced PCR-detected Mtb DNA for dilution series and Cohort A sputum versus untreated controls, suggesting non-intact Mtb is present before treatment-start. PEMAX enabled sequencing-based Mycobacterium-detection in 7/12 (58%) TB-positive sputa where no such reads otherwise occurred. In Cohort A, PMA- and PEMAX-treated versus untreated sputa had decreased α- and increased ß-diversities. In Cohort B, ß-diversity differences between timepoints were only detected with PEMAX. DNaseI had negligible effects. PMA and PEMAX (but not DNaseI) reduced extracellular DNA in PCR and improved pathogen detection by sequencing. PEMAX additionally detected chemotherapy-associated taxonomic changes that would otherwise be missed. Dyes enhance microbiome evaluations especially during chemotherapy.

Short-read full-length 16S rRNA amplicon sequencing for characterisation of the respiratory bacteriome of captive and free-ranging African elephants (Loxodonta africana).

Hypervariable region sequencing of the 16S ribosomal RNA (rRNA) gene plays a critical role in microbial ecology by offering insights into bacterial communities within specific niches. While providing valuable genus-level information, its reliance on data from targeted genetic regions limits its overall utility. Recent advances in sequencing technologies have enabled characterisation of the full-length 16S rRNA gene, enhancing species-level classification. Although current short-read platforms are cost-effective and precise, they lack full-length 16S rRNA amplicon sequencing capability. This study aimed to evaluate the feasibility of a modified 150 bp paired-end full-length 16S rRNA amplicon short-read sequencing technique on the Illumina iSeq 100 and 16S rRNA amplicon assembly workflow by utilising a standard mock microbial community and subsequently performing exploratory characterisation of captive (zoo) and free-ranging African elephant (Loxodonta africana) respiratory microbiota. Our findings demonstrate that, despite generating assembled amplicons averaging 869 bp in length, this sequencing technique provides taxonomic assignments consistent with the theoretical composition of the mock community and respiratory microbiota of other mammals. Tentative bacterial signatures, potentially representing distinct respiratory tract compartments (trunk and lower respiratory tract) were visually identified, necessitating further investigation to gain deeper insights into their implication for elephant physiology and health.

Mycobacterial genomic DNA from used Xpert MTB/RIF cartridges can be utilised for accurate second-line genotypic drug susceptibility testing and spoligotyping.

Xpert MTB/RIF (Xpert) is a widely-used test for tuberculosis (TB) and rifampicin-resistance. Second-line drug susceptibility testing (DST), which is recommended by policymakers, typically requires additional specimen collection that delays effective treatment initiation. We examined whether cartridge extract (CE) from used Xpert TB-positive cartridges was, without downstream DNA extraction or purification, suitable for both genotypic DST (MTBDRplus, MTBDRsl), which may permit patients to rapidly receive a XDR-TB diagnosis from a single specimen, and spoligotyping, which could facilitate routine genotyping. To determine the limit-of-detection and diagnostic accuracy, CEs from dilution series of drug-susceptible and -resistant bacilli were tested (MTBDRplus, MTBDRsl). Xpert TB-positive patient sputa CEs (n = 85) were tested (56 Xpert-rifampicin-susceptible, MTBDRplus and MTBDRsl; 29 Xpert-rifampicin-resistant, MTBDRsl). Spoligotyping was done on CEs from dilution series and patient sputa (n = 10). MTBDRplus had high non-valid result rates. MTBDRsl on CEs from dilutions ≥103CFU/ml (CT ≤ 24, >"low" Xpert semiquantitation category) was accurate, had low indeterminate rates and, on CE from sputa, highly concordant with MTBDRsl isolate results. CE spoligotyping results from dilutions ≥103CFU/ml and sputa were correct. MTBDRsl and spoligotyping on CE are thus highly feasible. These findings reduce the need for additional specimen collection and culture, for which capacity is limited in high-burden countries, and have implications for diagnostic laboratories and TB molecular epidemiology.