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Background. Persistent or recurrent pneumonia during antimicrobial therapy in patients receiving chemotherapy can be difficult to interpret. It may reflect persistence of the initial pathogen, inadequate antimicrobial exposure, noninfectious mimics, aspiration, superinfection or acquisition of a new respiratory pathogen. We report a case illustrating the clinical importance of repeat microbiological assessment when pneumonia persists or worsens despite apparently appropriate antimicrobial treatment. Case presentation. A man in his 80s with advanced classical Hodgkin lymphoma and chronic obstructive pulmonary disease developed pneumonia during chemotherapy. The initial sputum culture yielded cefepime-susceptible Klebsiella oxytoca. Cefepime produced transient defervescence, but oxygen requirements and radiographic abnormalities persisted, and fever recurred from day 18 with radiographic progression by day 19. Repeat sputum obtained on day 23 subsequently yielded extended-spectrum beta-lactamase-producing Klebsiella pneumoniae resistant to piperacillin/tazobactam and cefepime but susceptible to carbapenems. The isolate showed a positive string test, consistent with a hypermucoviscous phenotype. Doripenem was started after the resistant isolate was reported; however, the patient later developed progressive bilateral pulmonary infiltrates and respiratory failure and died. The interpretation of microbiological replacement remained presumptive because the organisms were recovered from expectorated sputum and molecular characterization was unavailable. Conclusion. This case highlights presumptive microbiological replacement as a clinically important consideration when pneumonia persists or worsens during chemotherapy. Repeat respiratory culture and updated susceptibility testing can materially alter antimicrobial management. A positive string test should be interpreted as a supplementary microbiological clue rather than proof of hypervirulence, metastatic infection or causality for death.

Klebsiella pneumoniae is a major cause of healthcare-associated and community-acquired infections, increasingly complicated by multidrug resistance (MDR) and carbapenem-resistant K. pneumoniae (CRKp). Phage-based approaches are attractive in such settings but rely on infrastructure and expertise, which are not widely available in low-resource laboratories. Here, we describe the isolation and preliminary characterization of two lytic K. pneumoniae phages, Zm_01 and Zm_02, from hospital sewer sludge in Lusaka, Zambia, using only routine microbiology equipment and simple adaptations of standard protocols. Clinical K. pneumoniae isolates, including MDR and carbapenem-resistant (meropenem-resistant) strains, were used for enrichment, double-agar overlay plaque assays, host-range screening and determination of efficiency of plating (EOP). Zm_01, isolated on a non-MDR but hypermucoviscous host, produced small, clear plaques and exhibited efficient plating on several MDR isolates, with EOP values higher on some MDR strains than on its original host. Zm_02, isolated on an MDR strain, formed large, clear plaques with halos, consistent with capsule-degrading activity. All work was performed without access to specialized equipment, such as a spectrophotometer, a shaker incubator or standard imaging equipment. These findings demonstrate that discovery and basic phenotypic characterization of therapeutically relevant K. pneumoniae phages are feasible in low-resource settings and provide a practical workflow that can underpin future functional and genomic studies, as well as the development of locally tailored phage-based interventions against MDR and CRKp infections.

Candida species have been attributed to causing ~647,000 deaths annually. Candida albicans has been identified by the WHO as a priority pathogen for targeted antifungal drug development. To this end, we have screened microbial strains from the public outreach project Swab and Send for anti-Candida activity. This process identified Pseudomonas sp. SS1954.14, which displayed potent activity against C. albicans, both on agar and in a cell-free supernatant assay. Whole-genome sequence analysis identified this strain as Pseudomonas monsensis. It contains several biosynthetic gene clusters, suggesting that it can produce hydrogen cyanide, lokisin, pyoverdine and colicin/carocin, which may contribute to the observed antifungal activity. However, an active compound was separated by preparative high-performance liquid chromatography and identified through high-resolution mass spectrometry as maculosin [cyclo(Pro-Tyr)]. This cyclic dipeptide has previously been found to possess antifungal activity, but the exact biosynthetic mechanism remains undetermined. Reporting this genome alongside the associated evidence of maculosin production represents a valuable resource for biosynthetic investigations.

Plasmodium vivax is a major cause of human malaria. It harbours Matryoshka RNA virus 1 (MaRNAV-1), a bi-segmented positive-sense RNA virus. MaRNAV-1 was first described in P. vivax and is now recognized as part of a wider group of Matryoshka viruses. These viruses also infect other haemosporidian parasites such as Leucocytozoon and Haemoproteus. The presence of MaRNAV-1 in African-origin human P. vivax, however, has not been clearly established. This study investigated whether MaRNAV-1 is present in public African-origin P. vivax transcriptomic datasets. Any viral sequences recovered were characterized using comparative genomic and phylogenetic analyses. A secondary in silico analysis targeted African-origin P. vivax RNA-seq runs from public repositories. Although the search covered Africa, only Ethiopian datasets could be confidently identified, retrieved and compiled at the time. After quality control and screening for MaRNAV-1 RNA-dependent RNA polymerase (RdRp) signals, three high-confidence runs were selected for further analysis. Reference-guided reconstruction, ORF prediction, blast-based validation and RdRp phylogenetic analysis were performed. MaRNAV-1 was identified in three Ethiopian P. vivax malaria transcriptomes. This was supported by strong segment-level mapping, near-complete coverage, high mean depth and minimal low-depth masking. The recovered genomes showed the expected bisegmented organization of MaRNAV-1. Segment I was highly conserved and encoded the canonical RdRp in all three consensus sequences. Segment II showed the conserved organization of two overlapping hypothetical ORFs in all three consensus sequences. Blast analyses confirmed close similarity to MaRNAV-1 reference sequences. Phylogenetic inference grouped the Ethiopian sequences within the broader P. vivax-associated MaRNAV-1 lineage, alongside other recognized MaRNAV lineages distinct from more divergent narna-like viruses. These findings provide genomic evidence for MaRNAV-1 in publicly available African-origin P. vivax transcriptomic datasets and add to the emerging evidence for the virus in the African malaria context.

Neoehrlichia mikurensis is an emerging tick-borne pathogen that is increasingly reported and can cause fever of unknown origin in immunocompromised individuals. We report a case of neoehrlichiosis occurring after B-cell-depleting treatment. Despite the availability of effective and inexpensive treatment, patients often experience significant care delays due to diagnostic challenges and insufficient awareness.

Isolated tuberculous abscess of the liver is an extremely rare clinical entity and represents a diagnostic challenge, as it can easily be confused with other hepatic lesions of infectious or tumoral origin. We report the case of a 67-year-old immunocompromised female patient with no known history of tuberculosis who was admitted for a liver abscess initially detected by abdominal computed tomography scan. Aspiration of the lesion revealed an infection involving Mycobacterium tuberculosis, confirmed by PCR analysis. The patient was treated with an appropriate antibiotic regimen of standard four-drug antituberculous therapy. Clinical and radiological follow-up showed progressive improvement, with complete recovery achieved after 6 months of treatment. This case highlights the importance of considering hepatic tuberculosis, particularly in endemic regions and in immunocompromised individuals.

Insertion sequences (ISs) are small, self-mobilizing DNA elements widespread across prokaryotic genomes, including chromosomes and plasmids. IS elements frequently co-localize with antimicrobial resistance (AMR) genes and mediate their mobilization, often as part of larger genomic structures that encompass multiple IS elements and antibiotic resistance genes. In this study, we employed Polymerase Chain Reaction (PCR) to amplify DNA sequences containing two copies of an IS26 element from two Escherichia coli ST131 isolates. While the respective PCRs generated products of the expected size, we also observed multiple amplicons of unexpected sizes, which could be misinterpreted as population heterogeneity attributed to IS mobilization. By extracting, re-amplifying and sequencing individual PCR products, we demonstrate that these amplicons of unexpected sizes were indeed artefact products generated during the PCR reaction, likely mediated by within-PCR recombination of the IS26 sequences. Furthermore, PCRs with equally oriented primers, each located close to an IS26 element, also generated artefact amplicons. This research highlights the limitations of using PCR to assess DNA sequences encoding multiple copies of an IS element and therefore, the presence of these genomic structures or the mobilization of the respective IS elements should not be assessed by diagnostic PCR alone but be corroborated with complementary techniques.

Osteoarticular tuberculosis (TB) is a rare but serious form of extrapulmonary TB that often presents with nonspecific symptoms, leading to delayed diagnosis and an increased risk of joint destruction. We report a case of knee TB in an immunocompetent patient, underscoring the diagnostic challenges and emphasizing the role of molecular techniques in the early management of this uncommon form of TB.

Antimicrobial resistance increasingly poses a significant global health challenge. New antimicrobials are desperately needed, driving the need to explore novel sources of natural products. Snake venom has shown promise due to the complex mixture of proteins and peptides which demonstrate therapeutic utility in other aspects of healthcare. In this study, we took an iterative approach to screen 14 snake venoms against Escherichia coli and Klebsiella pneumoniae and identify venom-derived proteins with diverse activity against E. coli. Six venoms exhibited significant near-complete inhibitory activity, and four were retained after dialysis, confirming that the inhibitory effect was due to high-molecular-weight components above 3.5 kDa. Fractionation of two active venoms from Naja mossambica and Naja subfulva revealed protein fractions which have either subtle or significant antimicrobial activity. SDS-PAGE and mass spectrometry identified phospholipase A₂ isoforms in N. mossambica venom fractions and neurotoxic proteins (long and short neurotoxins) in N. subfulva venom fractions as potential antimicrobials.

Background. Cervical screening with high-precision assays such as human papillomavirus (HPV) DNA testing is essential for the detection and treatment of precancerous lesions. HPV genotypes have different oncogenic potential and require different clinical management, illustrating the importance of extended genotyping. HPV quantification has demonstrated clinical relevance in both diagnosis and treatment. Objective. To develop a droplet digital PCR assay for the detection and quantification of 16 HPV genotypes, with comparison to a commercial test and validation on clinical samples. Methods. Primers and probes were designed to target the E6 region of 16 HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82. Each target was evaluated to assess performance and reliability using synthetic DNA constructs, quantified international reference standards and clinical screening samples (n=303) genotyped using the Seegene Anyplex II HR HPV Detection assay. Results. Each assay demonstrated high target specificity, without cross-reactivity observed among the HPV genotypes selected. Using international molecular standards, the assay reliably detected high-risk genotypes across serial dilutions, with detection down to the level of one international unit or genome equivalent per microlitre. When applied to clinical samples with and without a histological diagnosis of cervical intraepithelial neoplasia 2 or worse (CIN2+), the assay reliably detected key HPV genotypes, with the exception of 7 of 234 (3%) of HPV-positive samples, all of which exhibited very low viral load. Conclusion. Although previous studies have described digital PCR methods targeting HPV E6, they have typically focused on a limited number of genotypes. This study expands upon existing methodology by introducing a sensitive and specific method for detection and quantification of 16 high-risk and potentially high-risk HPV genotypes.