Etiological spectrum of persistent fever in the tropics and predictors of ubiquitous infections: a prospective four-country study with pooled analysis.

BACKGROUND: Persistent fever, defined as fever lasting for 7 days or more at first medical evaluation, has been hardly investigated as a separate clinical entity in the tropics. This study aimed at exploring the frequencies and diagnostic predictors of the ubiquitous priority (i.e., severe and treatable) infections causing persistent fever in the tropics. METHODS: In six different health settings across four countries in Africa and Asia (Sudan, Democratic Republic of Congo [DRC], Nepal, and Cambodia), consecutive patients aged 5 years or older with persistent fever were prospectively recruited from January 2013 to October 2014. Participants underwent a reference diagnostic workup targeting a pre-established list of 12 epidemiologically relevant priority infections (i.e., malaria, tuberculosis, HIV, enteric fever, leptospirosis, rickettsiosis, brucellosis, melioidosis, relapsing fever, visceral leishmaniasis, human African trypanosomiasis, amebic liver abscess). The likelihood ratios (LRs) of clinical and basic laboratory features were determined by pooling all cases of each identified ubiquitous infection (i.e., found in all countries). In addition, we assessed the diagnostic accuracy of five antibody-based rapid diagnostic tests (RDTs): Typhidot Rapid IgM, Test-itTM Typhoid IgM Lateral Flow Assay, and SD Bioline Salmonella typhi IgG/IgM for Salmonella Typhi infection, and Test-itTM Leptospira IgM Lateral Flow Assay and SD Bioline Leptospira IgG/IgM for leptospirosis. RESULTS: A total of 1922 patients (median age: 35 years; female: 51%) were enrolled (Sudan, n = 667; DRC, n = 300; Nepal, n = 577; Cambodia, n = 378). Ubiquitous priority infections were diagnosed in 452 (23.5%) participants and included malaria 8.0% (n = 154), tuberculosis 6.7% (n = 129), leptospirosis 4.0% (n = 77), rickettsiosis 2.3% (n = 44), enteric fever 1.8% (n = 34), and new HIV diagnosis 0.7% (n = 14). The other priority infections were limited to one or two countries. The only features with a positive LR ≥ 3 were diarrhea for enteric fever and elevated alanine aminotransferase level for enteric fever and rickettsiosis. Sensitivities ranged from 29 to 67% for the three RDTs targeting S. Typhi and were 9% and 16% for the two RDTs targeting leptospirosis. Specificities ranged from 86 to 99% for S. Typhi detecting RDTs and were 96% and 97% for leptospirosis RDTs. CONCLUSIONS: Leptospirosis, rickettsiosis, and enteric fever accounted each for a substantial proportion of the persistent fever caseload across all tropical areas, in addition to malaria, tuberculosis, and HIV. Very few discriminative features were however identified, and RDTs for leptospirosis and Salmonella Typhi infection performed poorly. Improved field diagnostics are urgently needed for these challenging infections. TRIAL REGISTRATION: NCT01766830 at ClinicalTrials.gov.

Blood culture surveillance in a secondary care hospital in Benin: epidemiology of bloodstream infection pathogens and antimicrobial resistance.

BACKGROUND: Although global surveillance of antimicrobial resistance (AMR) is considered key in the containment of AMR, data from low- and middle-income countries, especially from sub-Saharan Africa, are scarce. This study describes epidemiology of bloodstream infections and antimicrobial resistance rates in a secondary care hospital in Benin. METHODS: Blood cultures were sampled, according to predefined indications, in BacT/ALERT FA Plus and PF Plus (bioMérieux, Marcy-l'Etoile, France) blood culture bottles (BCB) in a district hospital (Boko hospital) and to a lesser extent in the University hospital of Parakou. These BCB were incubated for 7 days in a standard incubator and twice daily inspected for visual signs of growth. Isolates retrieved from the BCB were processed locally and later shipped to Belgium for reference identification [matrix-assisted laser desorption/ionization time-of-flight spectrometry (MALDI-TOF)] and antibiotic susceptibility testing (disk diffusion and E-tests). RESULTS: From October 2017 to February 2020, 3353 BCB were sampled, corresponding to 3140 blood cultures (212 cultures consisting of  > 1 BCB) and 3082 suspected bloodstream infection (BSI) episodes. Most of these cultures (n = 2471; 78.7%) were sampled in children < 15 years of age. Pathogens were recovered from 383 (12.4%) cultures, corresponding to 381 confirmed BSI. 340 of these pathogens were available and confirmed by reference identification. The most common pathogens were Klebsiella pneumoniae (n = 53; 15.6%), Salmonella Typhi (n = 52; 15.3%) and Staphylococcus aureus (n = 46; 13.5%). AMR rates were high among Enterobacterales, with resistance to third-generation cephalosporins in 77.6% of K. pneumoniae isolates (n = 58), 12.8% of Escherichia coli isolates (n = 49) and 70.5% of Enterobacter cloacae isolates (n = 44). Carbapenemase production was detected in 2 Escherichia coli and 2 Enterobacter cloacae isolates, all of which were of the New Delhi metallo-beta lactamase type. Methicillin resistance was present in 22.4% of S. aureus isolates (n = 49). CONCLUSION: Blood cultures were successfully implemented in a district hospital in Benin, especially among the pediatric patient population. Unexpectedly high rates of AMR among Gram-negative bacteria against commonly used antibiotics were found, demonstrating the clinical and scientific importance of clinical bacteriology laboratories at this level of care.

High frequency of Taenia solium antigen positivity in patients admitted for neurological disorders in the Rural Hospital of Mosango, Democratic Republic of Congo.

BACKGROUND: The epidemiology of human cysticercosis and neurocysticercosis, caused by the larval stage of the pork tapeworm Taenia solium, is not well known in the Democratic Republic of Congo (DRC). Within a multicenter etiological and diagnostic study conducted by the NIDIAG consortium ("Better Diagnosis for Neglected Infections") and investigating several challenging syndromes, we consecutively evaluated from 2012 to 2015 all patients older than 5 years presenting with neurological disorders (neurology cohort) and with fever > 7 days (persistent fever cohort) at the rural hospital of Mosango, province of Kwilu, DRC. In both cohorts, etiological diagnosis relied on a systematic set of reference laboratory assays and on pre-established clinical case definitions. No neuroimaging was available in the study hospital. In this study, we determined the frequency of T. solium infection in both cohorts and explored in the neurology cohort its association with specific neurological presentations and final etiological diagnoses. METHODS: We conducted a post-hoc descriptive and analytic study on cysticercosis in the neurology and persistent fever cohorts, based on the presence in serum samples of circulating T. solium antigen using the B158/B60 enzyme-linked immunosorbent assay (ELISA) and of cysticercosis IgG using the LDBIO Cysticercosis Western Blot IgG assay. RESULTS: For the neurology cohort, 340 samples (of 351 enrolled patients) were available for analysis (males: 46.8%; mean age: 38.9 years). T. solium antigen positivity was found in 43 participants (12.6%; 95% confidence interval [CI] 9.3-16.7%), including 9 of 60 (15%) patients with epilepsy. Among the 148 samples available from the persistent fever cohort (males: 39.9%; mean age: 19.9 years), 7 were positive in the T. solium antigen ELISA (4.7%; 95% CI 1.9-9.5%; P = 0.009 when compared to the neurology cohort). No significant association was found within the neurology cohort between positivity and clinical presentation or final diagnoses. Of note, the IgG antibody-detecting assay was found positive in only four (1.3%) of the participants of the neurology cohort and in none of the persistent fever cohort. CONCLUSIONS: T. solium antigen positivity was found in at least 10% of patients admitted with neurological disorders in the Kwilu province, DRC, with no specific pattern of presentation. Further neuroimaging studies should be used to confirm whether neurocysticercosis is prevalent in this region.

Promising application of monoclonal antibody against chikungunya virus E1-antigen across genotypes in immunochromatographic rapid diagnostic tests.

BACKGROUND: Three different genotypes of chikungunya virus (CHIKV) have been classified: East/Central/South African (ECSA), West African (WA), and Asian. Previously, a rapid immunochromatographic (IC) test detecting CHIKV E1-antigen showed high sensitivity for certain ECSA-genotype viruses, but this test showed poor performance against the Asian-genotype virus that is spreading in the American continents. We found that the reactivity of one monoclonal antibody (MAb) used in the IC rapid diagnostic test (RDT) is affected by a single amino acid substitution in E1. Therefore, we developed new MAbs that exhibited specific recognition of all three genotypes of CHIKV. METHODS: Using a combination of the newly generated MAbs, we developed a novel version of the IC RDT with improved sensitivity to Asian-genotype CHIKV. To evaluate the sensitivity, specificity, and cross-reactivity of the new version of the IC RDT, we first used CHIKV isolates and E1-pseudotyped lentiviral vectors. We then used clinical specimens obtained in Aruba in 2015 and in Bangladesh in 2017 for further evaluation of RDT sensitivity and specificity. Another alphavirus, sindbis virus (SINV), was used to test RDT cross-reactivity. RESULTS: The new version of the RDT detected Asian-genotype CHIKV at titers as low as 10^4 plaque-forming units per mL, a concentration that was below the limit of detection of the old version. The new RDT had sensitivity to the ECSA genotype that was comparable with that of the old version, yielding 92% (92 out of 100) sensitivity (95% confidence interval 85.0-95.9) and 100% (100 out of 100) specificity against a panel of 100 CHIKV-positive and 100 CHIKV-negative patient sera obtained in the 2017 outbreak in Bangladesh. CONCLUSIONS: Our newly developed CHIKV antigen-detecting RDT demonstrated high levels of sensitivity and lacked cross-reactivity against SINV. These results suggested that our new version of the CHIKV E1-antigen RDT is promising for use in areas in which the Asian and ECSA genotypes of CHIKV circulate. Further validation with large numbers of CHIKV-positive and -negative clinical samples is warranted. (323 words).

Salmonella Typhi From Blood Cultures in the Democratic Republic of the Congo: A 10-Year Surveillance.

BACKGROUND: This study gives an overview of a decade (2007-2017) of hospital-based Salmonella Typhi bloodstream infection (BSI) surveillance in the Democratic Republic of the Congo (DRC), at 4 main sampling sites. METHODS: Blood cultures were sampled in hospital-admitted patients with suspected BSI, according to standardized clinical indications. The results of the surveillance period 2015-2017 were compiled with those of previous surveillance periods (2007-2010 and 2011-2014). Whole genome sequencing of isolates with decreased ciprofloxacin susceptibility (DCS) was performed. RESULTS: Salmonella Typhi was isolated in 1.4% (531/37 388) and 10.3% (531/5177) of suspected and culture-confirmed BSI episodes, respectively. Salmonella Typhi ranked first among the BSI pathogens in adults (n = 220), but was mostly (n = 301 [56.7%]) isolated from children, of which 72.1% (217/301) and 31.6% (95/301) were <10 years and <5 years old, respectively. Multidrug resistance (MDR), DCS, and combined MDR/DCS were found in 38.3% (n = 180), 24.5% (n = 115), and 11.9% (n = 56) of 470 first isolates, respectively. MDR and DCS rates had increased since 2007, but remained stable during 2015-2017 with no geographical clustering at the province level. Most (91/93 [97.8%]) DCS isolates sequenced belonged to Genotyphi genotype 2.5.1, and gyr S83 was the most frequent DCS mutation (76/93 [81.7%]). Infections occurred perennially, but increased during the rainy season. CONCLUSIONS: Salmonella Typhi was a frequent cause of BSI in adults and children in DRC, with high rates of antibiotic resistance. Sustainable surveillance and implementation of vaccination are compelling.

Salmonella enterica serovar Typhi Producing CTX-M-15 Extended Spectrum ß-Lactamase in the Democratic Republic of the Congo.

We report a typhoid fever case with a Salmonella enterica serovar Typhi isolate showing extended spectrum ß-lactamase (ESBL) production in the Democratic Republic of the Congo. Whole genome sequencing revealed that the strain carried a plasmid-mediated CTX-M-15 ESBL gene and did not belong to the dominant H58 Salmonella Typhi clade.

Gambiense human african trypanosomiasis and immunological memory: effect on phenotypic lymphocyte profiles and humoral immunity.

In mice, experimental infection with Trypanosoma brucei causes decreased bone marrow B-cell development, abolished splenic B-cell maturation and loss of antibody mediated protection including vaccine induced memory responses. Nothing is known about this phenomenon in human African trypanosomiasis (HAT), but if occurring, it would imply the need of revaccination of HAT patients after therapy and abolish hope for a HAT vaccine. The effect of gambiense HAT on peripheral blood memory T- and B-cells and on innate and vaccine induced antibody levels was examined. The percentage of memory B- and T-cells was quantified in peripheral blood, prospectively collected in DR Congo from 117 Trypanosoma brucei gambiense infected HAT patients before and six months after treatment and 117 controls at the same time points. Antibodies against carbohydrate antigens on red blood cells and against measles were quantified. Before treatment, significantly higher percentages of memory B-cells, mainly T-independent memory B-cells, were observed in HAT patients compared to controls (CD20+CD27+IgM+, 13.0% versus 2.0%, p<0.001). The percentage of memory T-cells, mainly early effector/memory T-cells, was higher in HAT (CD3+CD45RO+CD27+, 19.4% versus 16.7%, p = 0.003). After treatment, the percentage of memory T-cells normalized, the percentage of memory B-cells did not. The median anti-red blood cell carbohydrate IgM level was one titer lower in HAT patients than in controls (p<0.004), and partially normalized after treatment. Anti-measles antibody concentrations were lower in HAT patients than in controls (medians of 1500 versus 2250 mIU/ml, p = 0.02), and remained so after treatment, but were above the cut-off level assumed to provide protection in 94.8% of HAT patients, before and after treatment (versus 98.3% of controls, p = 0.3). Although functionality of the B-cells was not verified, the results suggest that immunity was conserved in T.b. gambiense infected HAT patients and that B-cell dysfunction might not be that severe as in mouse models.

Invasive Salmonella Infections at Multiple Surveillance Sites in the Democratic Republic of the Congo, 2011-2014.

BACKGROUND: This study reports the microbiological landscape of Salmonella Typhi and invasive nontyphoidal Salmonella (iNTS) in the Democratic Republic of the Congo (DRC). METHODS: Blood cultures obtained from hospital-admitted patients suspected of bloodstream infection (BSI) in 4 of 11 provinces in DRC (Kinshasa, Bas-Congo, Equateur, and Orientale) were processed. Sampling had started in 2007; the results for the period 2011-2014 are reported. RESULTS: Salmonella Typhi and iNTS were cultured from 194 (1.4%) and 840 (5.9%), respectively, of 14,110 BSI episodes and ranked first among BSI pathogens in adults (65/300 [21.7%]) and children (783/1901 [41.2%]), respectively. A total of 948 of 1034 (91.7%) isolates were available for analysis (164 Salmonella Typhi and 784 iNTS). Salmonella Typhimurium and Salmonella Enteritidis represented 386 (49.2%) and 391 (49.9%), respectively, of iNTS isolates, fluctuating over time and geography and increasing during the rainy season. Adults accounted for <5% of iNTS BSI episodes. Children <5 years accounted for 20.3% of Salmonella Typhi BSI episodes. Among Salmonella Typhi, rates of multidrug resistance and decreased ciprofloxacin susceptibility (DCS) were 37.8% and 37.2%, respectively, and 18.3% displayed combined multidrug resistance and DCS; rates of azithromycin and ceftriaxone resistance were 0.6% and absent, respectively. Among NTS isolates, ≥80% (79.7% of Salmonella Enteritidis and 90.2% of Salmonella Typhimurium isolates) showed multidrug resistance, and <2.5% showed DCS. Combined extended-spectrum ß-lactamase production (blaTEM-1 gene) and azithromycin resistance was noted in 12.7% of Salmonella Typhimurium isolates, appearing in Bas-Congo from 2013 onward. CONCLUSIONS: Salmonella Typhi and NTS are major causes of BSI in DRC; their antimicrobial resistance is increasing.

SMS photograph-based external quality assessment of reading and interpretation of malaria rapid diagnostic tests in the Democratic Republic of the Congo.

BACKGROUND: The present External Quality Assessment (EQA) assessed reading and interpretation of malaria rapid diagnostic tests (RDTs) in the Democratic Republic of the Congo (DRC). METHODS: The EQA consisted of (i) 10 high-resolution printed photographs displaying cassettes with real-life results and multiple choice questions (MCQ) addressing individual health workers (HW), and (ii) a questionnaire on RDT use addressing the laboratory of health facilities (HF). Answers were transmitted through short message services (SMS). RESULTS: The EQA comprised 2344 HW and 1028 HF covering 10/11 provinces in DRC. Overall, median HW score (sum of correct answers on 10 MCQ photographs for each HW) was 9.0 (interquartile range 7.5 - 10); MCQ scores (the % of correct answers for a particular photograph) ranged from 54.8% to 91.6%. Most common errors were (i) reading or interpreting faint or weak line intensities as negative (3.3%, 7.2%, 24.3% and 29.1% for 4 MCQ photographs), (ii) failure to distinguish the correct Plasmodium species (3.4% to 7.0%), (iii) missing invalid test results (8.4% and 23.6%) and (iv) missing negative test results (10.0% and 12.4%). HW who were trained less than 12 months ago had best MCQ scores for 7/10 photographs as well as a significantly higher proportion of 10/10 scores, but absolute differences in MCQ scores were small. HW who had participated in a previous EQA performed significantly better for 4/10 photographs compared to those who had not. Except for two photographs, MCQ scores were comparable for all levels of the HF hierarchy and non-laboratory staff (HW from health posts) had similar performance as to laboratory staff. Main findings of the questionnaire were (i) use of other RDT products than recommended by the national malaria control programme (nearly 20% of participating HF), (ii) lack of training for a third (33.6%) of HF, (iii) high proportions (two-thirds, 66.5%) of HF reporting stock-outs. CONCLUSIONS: The present EQA revealed common errors in RDT reading and interpretation by HW in DRC. Performances of non-laboratory and laboratory staff were similar and dedicated training was shown to improve HW competence although to a moderate extent. Problems in supply, distribution and training of RDTs were detected.

Harmonization of malaria rapid diagnostic tests: best practices in labelling including instructions for use.

BACKGROUND: Rapid diagnostic tests (RDTs) largely account for the scale-up of malaria diagnosis in endemic settings. However, diversity in labelling including the instructions for use (IFU) limits their interchangeability and user-friendliness. Uniform, easy to follow and consistent labelling, aligned with international standards and appropriate for the level of the end user's education and training, is crucial but a consolidated resource of information regarding best practices for IFU and labelling of RDT devices, packaging and accessories is not available. METHODS: The Roll Back Malaria Partnership (RBM) commissioned the compilation of international standards and regulatory documents and published literature containing specifications and/or recommendations for RDT design, packaging and labelling of in vitro diagnostics (IVD) (which includes RDTs), complemented with a questionnaire based survey of RDT manufacturers and implementers. A summary of desirable RDT labelling characteristics was compiled, which was reviewed and discussed during a RBM Stakeholder consultation meeting and subsequently amended and refined by a dedicated task force consisting of country programme implementers, experts in RDT implementation, IVD regulatory experts and manufacturers. RESULTS: This process led to the development of consensus documents with a list of suggested terms and abbreviations as well as specifications for labelling of box, device packaging, cassettes, buffer bottle and accessories (lancets, alcohol swabs, transfer devices, desiccants). Emphasis was placed on durability (permanent printing or water-resistant labels), legibility (font size, letter type), comprehension (use of symbols) and ease of reference (e.g. place of labelling on the box or cassette packaging allowing quick oversight). A generic IFU template was developed, comprising background information, a template for procedure and reading/interpretation, a selection of appropriate references and a symbol key of internationally recognized symbols together with suggestions about appropriate lay-out, style and readability. CONCLUSIONS: The present document together with its additional files compiled proposes best practices in labelling and IFU for malaria RDTs. It is expected that compliance with these best practices will increase harmonization among the different malaria RDT products available on the market and improve their user-friendliness.

A genomic appraisal of invasive Salmonella Typhimurium and associated antibiotic resistance in sub-Saharan Africa.

Invasive non-typhoidal Salmonella (iNTS) disease manifesting as bloodstream infection with high mortality is responsible for a huge public health burden in sub-Saharan Africa. Salmonella enterica serovar Typhimurium (S. Typhimurium) is the main cause of iNTS disease in Africa. By analysing whole genome sequence data from 1303 S. Typhimurium isolates originating from 19 African countries and isolated between 1979 and 2017, here we show a thorough scaled appraisal of the population structure of iNTS disease caused by S. Typhimurium across many of Africa's most impacted countries. At least six invasive S. Typhimurium clades have already emerged, with ST313 lineage 2 or ST313-L2 driving the current pandemic. ST313-L2 likely emerged in the Democratic Republic of Congo around 1980 and further spread in the mid 1990s. We observed plasmid-borne as well as chromosomally encoded fluoroquinolone resistance underlying emergences of extensive-drug and pan-drug resistance. Our work provides an overview of the evolution of invasive S. Typhimurium disease, and can be exploited to target control measures.

Assessment of desiccants and their instructions for use in rapid diagnostic tests.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are protected from humidity-caused degradation by a desiccant added to the device packaging. The present study assessed malaria RDT products for the availability, type and design of desiccants and their information supplied in the instructions for use (IFU). METHODS: Criteria were based on recommendations of the World Health Organization (WHO), the European Community (CE) and own observations. Silica gel sachets were defined as self-indicating (all beads coated with a humidity indicator that changes colour upon saturation), partial-indicating (part of beads coated) and non-indicating (none of the beads coated). Indicating silica gel sachets were individually assessed for humidity saturation and (in case of partial-indicating silica gels) for the presence of indicating beads. RESULTS: Fifty malaria RDT products from 25 manufacturers were assessed, 14 (28%) products were listed by the "Global Fund Quality Assurance Policy" and 31 (62%) were CE-marked. All but one product contained a desiccant, mostly (47/50, 94%) silica gel. Twenty (40%) RDT products (one with no desiccant and 19 with non-indicating desiccant) did not meet the WHO guidelines recommending indicating desiccant. All RDT products with self- or partial-indicating silica gel (n = 22 and 8 respectively) contained the toxic cobalt dichloride as humidity indicator. Colour change indicating humidity saturation was observed for 8/16 RDT products, at a median incidence of 0.8% (range 0.05%-4.6%) of sachets inspected. In all RDTs with partial-indicating silica gel, sachets with no colour indicating beads were found (median proportion 13.5% (0.6%-17.8%) per product) and additional light was needed to assess the humidity colour. Less than half (14/30, 47%) IFUs of RDT products with indicating desiccants mentioned to check the humidity saturation before using the test. Information on properties, safety hazards and disposal of the desiccant was not included in any of the IFUs. There were no differences between Global Fund-listed and CE marked RDT products compared to those which were not. Similar findings were noted for a panel of 11 HIV RDTs that was assessed with the same checklist as the malaria RDTs. CONCLUSION: RDTs showed shortcomings in desiccant type and information supplied in the IFU.

Pilot Testing of the "Turbidimeter", a Simple, Universal Reader Intended to Complement and Enhance Bacterial Growth Detection in Manual Blood Culture Systems in Low-Resource Settings.

Bloodstream infections and antimicrobial resistance are an increasing problem in low-income countries. There is a clear need for adapted diagnostic tools. To address this need, we developed a simple, universal reader prototype that detects bacterial growth in blood culture bottles. Our "turbidimeter" evaluates bacterial growth, based on the turbidity of the broth and the color change of the colorimetric CO2 indicator in commercially available blood culture bottles. A total of 60 measurements were performed using 10 relevant microbial species, spiked in horse blood, to compare the turbidimeter's performance with that of an automatic reference system. The turbidimeter was able to detect growth in all but one of the spiked blood culture bottles. In the majority (7/10) of the species tested, time-to-detection of the turbidimeter was shown to be non-inferior to the reference automated time-to-detection. This was, however, only the case when both the turbidity and color change in the colorimetric CO2-indicator were used to evaluate growth. We could not demonstrate the non-inferiority of the turbidity measurement alone. Overall, the turbidimeter performed well, but we also identified some improvements that will be implemented in the next version of the prototype.

Accuracy of C-reactive Protein and Procalcitonin for Diagnosing Bacterial Infections Among Subjects With Persistent Fever in the Tropics.

Background: In low-resource settings, inflammatory biomarkers can help identify patients with acute febrile illness who do not require antibiotics. Their use has not been studied in persistent fever (defined as fever lasting for ≥7 days at presentation). Methods: C-reactive protein (CRP) and procalcitonin (PCT) levels were measured in stored serum samples of patients with persistent fever prospectively enrolled in Cambodia, the Democratic Republic of Congo, Nepal, and Sudan. Diagnostic accuracy was assessed for identifying all bacterial infections and the subcategory of severe infections judged to require immediate antibiotics. Results: Among 1838 participants, CRP and PCT levels were determined in 1777 (96.7%) and 1711 (93.1%) samples, respectively, while white blood cell (WBC) count was available for 1762 (95.9%). Areas under the receiver operating characteristic curve for bacterial infections were higher for CRP (0.669) and WBC count (0.651) as compared with PCT (0.600; P <.001). Sensitivity for overall and severe bacterial infections was 76.3% (469/615) and 88.2% (194/220) for CRP >10 mg/L, 62.4% (380/609) and 76.8% (169/220) for PCT >0.1 µg/L, and 30.5% (184/604) and 43.7% (94/215) for WBC >11 000/µL, respectively. Initial CRP level was <10 mg/L in 45% of the participants who received antibiotics at first presentation. Conclusions: In patients with persistent fever, CRP and PCT showed higher sensitivity for bacterial infections than WBC count, applying commonly used cutoffs for normal values. A normal CRP value excluded the vast majority of severe infections and could therefore assist in deciding whether to withhold empiric antibiotics after cautious clinical assessment.

Epidemiological cut-off value and antibiotic susceptibility test methods for azithromycin in a collection of multi-country invasive non-typhoidal Salmonella.

OBJECTIVE: Azithromycin is an alternative to treat invasive non-typhoidal Salmonella (iNTS) infections. We determined its epidemiological cut-off (ECOFF) and compared azithromycin susceptibility testing methods for iNTS. METHODS: We used EUCAST ECOFFinder to determine the minimum inhibitory concentrations (MIC; obtained by broth microdilution) ECOFF and corresponding disk zone diameters of 515 iNTS from blood cultures in Democratic Republic of Congo, Burkina Faso, Rwanda, and Cambodia. Transferable resistance mechanisms were determined by polymerase chain reaction. We compared azithromycin susceptibility testing by semi-automated broth microdilution (customized Sensititre panel; reference), agar dilution, gradient tests (bioMérieux, Liofilchem, HiMedia; read at 80% (MIC80%) and 100% inhibition (MIC100%)), and disk diffusion (Rosco, Oxoid, BD, Liofilchem) for 161 wild- and 198 non-wild-type iNTS. RESULTS: Azithromycin MIC ECOFF was 16 mg/L corresponding to a 12 mm zone diameter; mphA was detected in 192/197 non-wild- and 0/47 wild-type iNTS. Categorical agreement was excellent (≥98%) for all methods. Essential agreement was very good for agar dilution (>90%) but moderate for gradient tests (MIC80%: 52% to 71% and MIC100%: 72% to 91%). Repeatability was good for all methods/brands. Interreader agreement was high for broth microdilution and agar dilution (all ≤1 twofold dilution difference) and disk diffusion (>96% ≤3 mm difference) but lower for gradient tests (MIC80% & MIC100%: 83% to 94% ≤1 twofold dilution difference). DISCUSSION: Azithromycin ECOFF of iNTS was 16 mg/L, i.e. equal to Salmonella Typhi. Disk diffusion is an accurate, precise, and user-friendly alternative for agar dilution and broth microdilution. Reading gradient tests at 100% instead of 80% inhibition improved accuracy and precision.

Direct association between rainfall and non-typhoidal Salmonella bloodstream infections in hospital-admitted children in the Democratic Republic of Congo.

Non-typhoidal Salmonella (NTS) ranks first among causes of bloodstream infection in children under five years old in the Democratic Republic of Congo and has a case fatality rate of 15%. Main host-associated risk factors are Plasmodium falciparum malaria, anemia and malnutrition. NTS transmission in sub-Saharan Africa is poorly understood. NTS bloodstream infections mostly occur during the rainy season, which may reflect seasonal variation in either environmental transmission or host susceptibility. We hypothesized that environment- and host-associated factors contribute independently to the seasonal variation in NTS bloodstream infections in children under five years old admitted to Kisantu referral hospital in 2013-2019. We used remotely sensed rainfall and temperature data as proxies for environmental factors and hospital data for host-associated factors. We used principal component analysis to disentangle the interrelated environment- and host-associated factors. With timeseries regression, we demonstrated a direct association between rainfall and NTS variation, independent of host-associated factors. While the latter explained 17.5% of NTS variation, rainfall explained an additional 9%. The direct association with rainfall points to environmental NTS transmission, which should be explored by environmental sampling studies. Environmental and climate change may increase NTS transmission directly or via host susceptibility, which highlights the importance of preventive public health interventions.

Culture media for clinical bacteriology in low- and middle-income countries: challenges, best practices for preparation and recommendations for improved access.

BACKGROUND: Culture media are fundamental in clinical microbiology. In laboratories in low- and middle-income countries (LMICs), they are mostly prepared in-house, which is challenging. OBJECTIVES: This narrative review describes challenges related to culture media in LMICs, compiles best practices for in-house media preparation, gives recommendations to improve access to quality-assured culture media products in LMICs and formulates outstanding questions for further research. SOURCES: Scientific literature was searched using PubMed and predefined MeSH terms. In addition, grey literature was screened, including manufacturer's websites and manuals as well as microbiology textbooks. CONTENT: Bacteriology laboratories in LMICs often face challenges at multiple levels: lack of clean water and uninterrupted power supply, high environmental temperatures and humidity, dust, inexperienced and poorly trained staff, and a variable supply of consumables (often of poor quality). To deal with this at a base level, one should be very careful in selecting culture media. It is recommended to look for products supported by the national reference laboratory that are being distributed by an in-country supplier. Correct storage is key, as is appropriate preparation and waste management. Centralized media acquisition has been advocated for LMICs, a role that can be taken up by the national reference laboratories, next to guidance and support of the local laboratories. In addition, there is an important role in tropicalization and customization of culture media formulations for private in vitro diagnostic manufacturers, who are often still unfamiliar with the LMIC market and the plethora of bacteriology products. IMPLICATION: The present narrative review will assist clinical microbiology laboratories in LMICs to establish best practices for handling culture media by defining quality, regulatory and research paths.

Onchocerciasis Prevalence among Persons with Epilepsy in an Onchocerciasis Hypo-Endemic Area in the Democratic Republic of Congo: A Cross-Sectional Study.

A high epilepsy prevalence has been reported in onchocerciasis meso- and hyper-endemic regions in sub-Saharan Africa, including in the Democratic Republic of Congo (DRC). We investigated whether onchocerciasis-associated epilepsy can also be suspected in onchocerciasis hypo-endemic regions. Stored serum samples from 342 patients admitted with recent onset neurological symptoms admitted to Mosango general hospital, in the Kwilu province, DRC, between 2012 and 2015 were screened for onchocerciasis (OV16) antibodies by ELISA and Taenia solium antigen (using an in-house B158/B60 antigen test). Eighty-one (23.7%; 95% CI 19.5-28.5%) of these samples were positive for OV16 antibodies and 43/340 (12.6%; 95% CI 9.5-16.6%) were positive for T. solium antigen. Of the 58 persons clinically diagnosed with late onset epilepsy of unknown etiology, 19 (32.8%) were OV16 positive and nine (16%) T. solium antigen positive. In total, 16 persons with epilepsy were OV16 positive and T. solium negative, of whom 12 (75%) were between the ages seven to 31 years old, an age rage in which onchocerciasis-associated epilepsy is observed. Our study suggests that in onchocerciasis hypo-endemic areas, in T. solium antigen negative persons with epilepsy, onchocerciasis should be considered as a potential trigger of epilepsy.

Antibiotic use prior to seeking medical care in patients with persistent fever: a cross-sectional study in four low- and middle-income countries.

OBJECTIVES: Community-level antibiotic use contributes to antimicrobial resistance, but is rarely monitored as part of efforts to optimize antibiotic use in low- and middle-income countries (LMICs). We investigated antibiotic use in the 4 weeks before study inclusion for persistent fever. METHODS: The NIDIAG-Fever (Neglected Infectious diseases DIAGnosis-Fever) study investigated aetiologies of infections in patients ≥5 years old with fever ≥1 week in six healthcare facilities in Cambodia, the Democratic Republic of the Congo (DRC), Nepal, and Sudan. In the present nested cross-sectional study, we describe prevalence and choice of antibiotics before and at study inclusion, applying the Access/Watch/Reserve (AWaRe) classification of the WHO List of Essential Medicines. Factors associated with prior antibiotic use were analysed. RESULTS: Of 1939 participants, 428 (22.1%) reported the prior use of one or more antibiotics, ranging from 6.3% (24/382, Cambodia) to 35.5% (207/583, Nepal). Of 545 reported antibiotics, the most frequent were Watch group antibiotics (351/545, 64.4%), ranging from 23.6% (DRC) to 82.1% (Nepal). Parenteral administration ranged from 5.9% to 69.6% between study sites. Antibiotic use was most frequent among young patients (5-17 years of age; risk ratio 1.42, 95%CI 1.19-1.71) and men (RR 1.29; 95%CI 1.09-1.53). No association was found with specific symptoms. Of 555 antibiotics started before study inclusion, 275 (49.5%) were discontinued at study inclusion. CONCLUSIONS: Watch antibiotics were frequently used, and discontinued upon study inclusion. The antibiotic use frequency and choice varied importantly between LMICs. Data on local antibiotic use are essential to guide efforts to optimize antibiotic use in LMICs, should not be restricted to hospitals, and need to take local healthcare utilization into account.