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Background: Fluoroquinolones have been, and continue to be, routinely used for treatment of many bacterial infections. In recent years, most parts of the world have reported an increasing trend of fluoroquinolone resistant (FQR) Gram-negative bacteria. Methods: A cross-sectional study was conducted between March 2017 and July 2018 among children admitted due to fever to referral hospitals in Dar es Salaam, Tanzania. Rectal swabs were used to screen for carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE). ESBL-PE isolates were tested for quinolone resistance by disk diffusion method. Randomly selected fluroquinolone resistant isolates were characterized by using whole genome sequencing. Results: A total of 142 ESBL-PE archived isolates were tested for fluoroquinolone resistance. Overall phenotypic resistance to ciprofloxacin, levofloxacin and moxifloxacin was found in 68% (97/142). The highest resistance rate was seen among Citrobacterspp. (100%, 5/5), followed by Klebsiella.pneumoniae (76.1%; 35/46), Escherichiacoli (65.6%; 42/64) and Enterobacter spp. (31.9%; 15/47). Whole genome sequencing (WGS) was performed on 42 fluoroquinolone resistant-ESBL producing isolates and revealed that 38/42; or 90.5%, of the isolates carried one or more plasmid mediated quinolone resistance (PMQR) genes. The most frequent PMQR genes were aac(6')-lb-cr (74%; 31/42), followed by qnrB1 (40%; 17/42), oqx,qnrB6 and qnS1. Chromosomal mutations in gyrA, parC and parE were detected among 19/42 isolates, and all were in E.coli. Most of the E. coli isolates (17/20) had high MIC values of > 32 µg/ml for fluoroquinolones. In these strains, multiple chromosomal mutations were detected, and all except three strains had additional PMQR genes. Sequence types, ST131 and ST617 predominated among E.coli isolates, while ST607 was more common out of 12 sequence types detected among the K.pneumoniae. Fluoroquinolone resistance genes were mostly associated with the IncF plasmids. Conclusion: The ESBL-PE isolates showed high rates of phenotypic resistance towards fluoroquinolones likely mediated by both chromosomal mutations and PMQR genes. Chromosomal mutations with or without the presence of PMQR were associated with high MIC values in these bacteria strains. We also found a diversity of PMQR genes, sequence types, virulence genes, and plasmid located antimicrobial resistance (AMR) genes towards other antimicrobial agents. [ABSTRACT FROM AUTHOR]

Background: Over one million yearly deaths are attributable to Streptococcus pneumoniae and people living with HIV are particularly vulnerable. Emerging penicillin non-susceptible Streptococcus pneumoniae (PNSP) challenges therapy of pneumococcal disease. The aim of this study was to determine the mechanisms of antibiotic resistance among PNSP isolates by next generation sequencing. Methods: We assessed 26 PNSP isolates obtained from the nasopharynx from 537 healthy human immunodeficiency virus (HIV) infected adults in Dar es Salaam, Tanzania, participating in the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890, registered on 23rd March, 2017). Next generation whole genome sequencing on the Illumina platform was used to identify mechanisms of resistance to antibiotics among PNSP. Results: Fifty percent (13/26) of PNSP were resistant to erythromycin, of these 54% (7/13) and 46% (6/13) had MLSB phenotype and M phenotype respectively. All erythromycin resistant PNSP carried macrolide resistance genes; six isolates had mef(A)-msr(D), five isolates had both erm(B) and mef(A)-msr(D) while two isolates carried erm(B) alone. Isolates harboring the erm(B) gene had increased MIC (> 256 µg/mL) towards macrolides, compared to isolates without erm(B) gene (MIC 4-12 µg/mL) p < 0.001. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, the prevalence of azithromycin resistance was overestimated compared to genetic correlates. Tetracycline resistance was detected in 13/26 (50%) of PNSP and all the 13 isolates harbored the tet(M) gene. All isolates carrying the tet(M) gene and 11/13 isolates with macrolide resistance genes were associated with the mobile genetic element Tn6009 transposon family. Of 26 PNSP isolates, serotype 3 was the most common (6/26), and sequence type ST271 accounted for 15% (4/26). Serotypes 3 and 19 displayed high-level macrolide resistance and frequently carried both macrolide and tetracycline resistance genes. Conclusion: The erm(B) and mef(A)-msr(D) were common genes conferring resistance to MLSB in PNSP. Resistance to tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon. [ABSTRACT FROM AUTHOR]

Background We aimed to characterise the genetic determinants and context of two meropenem-resistant clinical isolates of Acinetobacter baumannii isolated from children hospitalised with bloodstream infections in Dar es Salaam, Tanzania. Methods Antimicrobial susceptibility was determined by disc diffusion E-test and broth microdilution. Genomes were completed using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing reads and characterisation of the genetic context of resistance genes, multi-locus sequence types (STs) and phylogenetic analysis was determined bioinformatically. Results Twelve A. baumannii were isolated from 2226 blood cultures, two of which were meropenem-resistant. The two meropenem-resistant isolates, belonging to distinct STs, ST374 and ST239, were found to harbour bla NDM-1, which was chromosomally located in isolate DT0544 and plasmid-located in isolate DT01139. The genetic environment of bla NDM-1 shows the association of insertion sequence ISAba 125 with bla NDM-1 in both isolates. Both isolates also harboured genes conferring resistance to other β-lactams, aminoglycosides and cotrimoxazole. Conclusions This is the first report of New Delhi metallo-β-lactamase-producing isolates of A. baumannii from Tanzania. The genetic context of bla NDM-1 provides further evidence of the importance of ISAba 125 in the spread of bla NDM-1 in A. baumannii. Local surveillance should be strengthened to keep clinicians updated on the incidence of these and other multidrug-resistant and difficult-to-treat bacteria. [ABSTRACT FROM AUTHOR]

Difficult-to-treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are of concern in people living with HIV infection as they are more vulnerable to infection. We aimed to identify molecular characteristics of MRSA colonizing newly diagnosed HIV-infected adults in Tanzania. Individuals newly diagnosed with HIV infection were recruited in Dar es Salaam, Tanzania, from April 2017 to May 2018, as part of the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890). Nasal/nasopharyngeal isolates of Staphylococcus aureus were susceptibility tested by disk diffusion method, and cefoxitin-resistant isolates were characterized by short-reads whole genome sequencing. Four percent (22/537) of patients carried MRSA in the nose/nasopharynx. MRSA isolates were frequently resistant towards gentamicin (95%), ciprofloxacin (91%), and erythromycin (82%) but less often towards trimethoprim-sulfamethoxazole (9%). Seventy-three percent had inducible clindamycin resistance. Erythromycin-resistant isolates harbored ermC (15/18) and LmrS (3/18) resistance genes. Ciprofloxacin resistance was mediated by mutations of the quinolone resistance-determining region (QRDR) sequence in the gyrA (S84L) and parC (S80Y) genes. All isolates belonged to the CC8 and ST8-SCCmecIV MRSA clone. Ninety-five percent of the MRSA isolates were spa-type t1476, and one exhibited spa-type t064. All isolates were negative for Panton-Valentine leucocidin (PVL) and arginine catabolic mobile element (ACME) type 1. All ST8-SCCmecIV-spa-t1476 MRSA clones from Tanzania were unrelated to the globally successful USA300 clone. Carriage of ST8 MRSA (non-USA300) was common among newly diagnosed HIV-infected adults in Tanzania. Frequent co-resistance to non-beta lactam antibiotics limits therapeutic options when infection occurs. [ABSTRACT FROM AUTHOR]

Background: Febrile illness is the commonest cause of hospitalization in children <5 years in sub-Saharan Africa, and bacterial bloodstream infections and malaria are major causes of death. Methods: From March 2017 to July 2018, we enrolled 2,226 children aged 0–5 years hospitalized due to fever in four major public hospitals of Dar es Salaam, namely, Amana, Temeke, and Mwananyamala Regional Hospitals and Muhimbili National Hospital. We recorded social demographic and clinical data, and we performed blood-culture and HIV-antibody testing. We used qPCR to quantify Plasmodium falciparum parasitaemia and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) to identify bacterial isolates. Disk diffusion method was used for antimicrobial susceptibility testing. Results: Nineteen percent of the children (426/2,226) had pathogens detected from blood. Eleven percent (236/2,226) of the children had bacteraemia/fungaemia and 10% (204/2,063) had P. falciparum malaria. Ten children had concomitant malaria and bacteraemia. Gram-negative bacteria (64%) were more frequent than Gram-positive (32%) and fungi (4%). Over 50% of Gram-negative bacteria were extended-spectrum beta-lactamase (ESBL) producers and multidrug resistant. Methicillin resistant Staphylococcus aureus (MRSA) was found in 11/42 (26.2%). The most severe form of clinical malaria was associated with high parasitaemia (>four million genomes/μL) of P. falciparum in plasma. Overall, in-hospital death was 4% (89/2,146), and it was higher in children with bacteraemia (8%, 18/227) than malaria (2%, 4/194, p = 0.007). Risk factors for death were bacteraemia (p = 0.03), unconsciousness at admission (p < 0.001), and admission at a tertiary hospital (p = 0.003). Conclusion: Compared to previous studies in this region, our study showed a reduction in malaria prevalence, a decrease in in-hospital mortality, and an increase in antimicrobial resistance (AMR) including ESBLs and multidrug resistance. An increase of AMR highlights the importance of continued strengthening of diagnostic capability and antimicrobial stewardship programs. We also found malaria and bacteraemia contributed equally in causing febrile illness, but bacteraemia caused higher in-hospital death. The most severe form of clinical malaria was associated with P. falciparum parasitaemia. [ABSTRACT FROM AUTHOR]

Background: Faecal carriage of ESBL-producing bacteria is a potential risk for transmission and infection. Little is known about faecal carriage of antibiotic resistance in Tanzania. This study aimed to investigate the prevalence of faecal carriage of ESBL-producing Enterobacteriaceae and to identify risk factors for carriage among young children in Tanzania. Methodology/Principal Findings: From August 2010 to July 2011, children below 2 years of age were recruited in Dar es Salaam, including healthy community children (n = 250) and children hospitalized due to diarrhoea (n = 250) or other diseases (n = 103). ChromID ESBL agar and ChromID CARBA SMART agar were used for screening. Antimicrobial susceptibility testing was performed by the disk diffusion method. ESBL genotypes were identified by Real-Time PCR and sequencing. The overall prevalence of ESBL carriage was 34.3% (207/ 603). The prevalence of ESBL carriage was significantly higher among hospitalized children (50.4%), compared to community children (11.6%; P < 0.001; OR = 7.75; 95% CI: 4.99–12.03). We found high prevalence of Multidrug-resistance (94%) among Escherichia coli and Klebsiella pneumoniae isolates. No resistance to carbapenems was detected. For the majority of isolates (94.7%) we detected a blaCTX-M-15-like gene. In addition, the plasmid mediated AmpC beta-lactamase CMY-2 was detected for the first time in Tanzania. ESBL prevalence was significantly higher among HIV positive (89.7%) than HIV negative (16.9%) children (P = 0.001; OR = 9.99; 95% CI: 2.52–39.57). Use of antibiotics during the past 14 days and age below 1 year was also associated with ESBL carriage. Conclusions/Significance: We report a high rate of faecal carriage of ESBL-producing Enterobacteriaceae among children below 2 years of age in Tanzania, particularly those with HIV-infection. Resistance to a majority of the available antimicrobials commonly used for children in Tanzania leaves few treatment options for infections when caused by these bacteria. [ABSTRACT FROM AUTHOR]

Background Asymptomatic malaria parasitaemia has been reported in areas with high malaria transmission. It may serve as a reservoir for continued transmission, and furthermore complicates diagnostics, as not all individuals with a positive malaria test are necessarily ill due to malaria, although they may present with malaria-like symptoms. Asymptomatic malaria increases with age as immunity to malaria gradually develops. As mortality and morbidity of malaria is higher among younger children it is important to know the prevalence of asymptomatic malaria parasitaemia in this population in order to interpret laboratory results for malaria correctly. Methods A total of 108 children that had neither been treated for malaria nor had a fever the previous four weeks were recruited consecutively at a maternal and child health clinic (MCHC) in Dar es Salaam, Tanzania. A rapid diagnostic test (RDT) for malaria and dried blood spot (DBS) on filter paper were taken from each child. Social and clinical data were recorded. DNA was extracted from the DBS of study participants by a method using InstaGeneTM matrix. PCR targeting the Plasmodium mitochondrial genome was performed on all samples. Results Median age was 4.6 months (range 0.5-38). All the RDTs were negative. PCR was negative for all study subjects. Conclusion The study suggests that asymptomatic malaria may not be present in apparently healthy children up to the age of three years in Dar es Salaam, Tanzania. However, because of the small sample size and low median age of the study population, the findings cannot be generalized. Larger studies, including higher age groups, need to be done to clarify whether asymptomatic malaria parasitaemia is present in the general population in the Dar es Salaam area. [ABSTRACT FROM AUTHOR]

Background: Malaria is a major cause of paediatric morbidity and mortality. As no clinical features clearly differentiate malaria from other febrile illnesses, and malaria diagnosis is challenged by often lacking laboratory equipment and expertise, overdiagnosis and overtreatment is common. Methods: Children admitted with fever at the general paediatric wards at Muhimbili National Hospital (MNH), Dar es Salaam, Tanzania from January to June 2009 were recruited consecutively and prospectively. Demographic and clinical features were registered. Routine thick blood smear microscopy at MNH was compared to results of subsequent thin blood smear microscopy, and rapid diagnostics tests (RDTs). Genus-specific PCR of Plasmodium mitochondrial DNA was performed on DNA extracted from whole blood and species-specific PCR was done on positive samples. Results: Among 304 included children, 62.6% had received anti-malarials during the last four weeks prior to admission and 65.1% during the hospital stay. Routine thick blood smears, research blood smears, PCR and RDT detected malaria in 13.2%, 6.6%, 25.0% and 13.5%, respectively. Positive routine microscopy was confirmed in only 43% (17/40), 45% (18/40) and 53% (21/40), by research microscopy, RDTs and PCR, respectively. Eighteen percent (56/304) had positive PCR but negative research microscopy. Reported low parasitaemia on routine microscopy was associated with negative research blood slide and PCR. RDT-positive cases were associated with signs of severe malaria. Palmar pallor, low haemoglobin and low platelet count were significantly associated with positive PCR, research microscopy and RDT. Conclusions: The true morbidity attributable to malaria in the study population remains uncertain due to the discrepancies in results among the diagnostic methods. The current routine microscopy appears to result in overdiagnosis of malaria and, consequently, overuse of anti-malarials. Conversely, children with a false positive malaria diagnosis may die because they do not receive treatment for the true cause of their illness. RDTs appear to have the potential to improve routine diagnostics, but the clinical implication of the many RDT-negative, PCR-positive samples needs to be elucidated. [ABSTRACT FROM AUTHOR]

This study investigated and reports norovirus diarrhoea, genetic diversity and associated clinical symptoms, HIV status and seasonality in a paediatric population of Tanzania. Stool specimens and demographic/clinical information, were prospectively collected from 705 hospitalised children with diarrhoea (cases) and 561 children without diarrhoea (controls) between 2010 and 2011. Norovirus detection was done by real-time RT-PCR. Genotype was determined using Gel-based and real time RT-PCR methods and sequencing targeting the polymerase and the capsid region respectively. Norovirus was detected in 14.3%, 181/1266 children. The prevalence of norovirus was significantly higher in cases (18.3%, 129/705) than in controls, (9.2%, 52/561), P < 0.05. Except for one child who had double infection with GI and GII all 129 cases had GII. Among controls, 23.1% had GI and 76.9% had GII. Norovirus GII.4 was significantly more prevalent in cases 87.9% than in controls 56.5%. Other genotypes detected in both cases and controls were GII.21, GII.16 and GII.g. The highest numbers of norovirus were detected in April 2011. The number of norovirus detected was significantly higher during the first than second year of life (109/540, 20.2% vs. 20/165, 12.1%). The prevalence of norovirus in HIV-positive and negative children was (21.2%, 7/33) and (10.3%, 40/390, P = 0.05) respectively, regardless of diarrhoea symptoms. No significant difference in gender, parent’s level of education or nutritional status with norovirus infection was observed within cases or controls. This study confirms the significant role of norovirus infection, especially GII.4 in diarrhoeic children who need hospitalisation and adds knowledge on norovirus epidemiology in the African region. [ABSTRACT FROM AUTHOR]

Colonization in HIV-infected populations with extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) is particularly worrisome in low-income settings. This study describes the prevalence of ESBL-PE carriage and associated risk factors among newly HIV-diagnosed adults in a community setting in Tanzania. A total of 595 newly diagnosed HIV adults with a median age of 35 years with interquartile range (IQR) 29–42 years and a median CD4 count of 492 cells/μL (IQR 390–666 cells/μL) were recruited. Among these, 194/595 (32.6%, 95% confidence interval [CI] 28.9–36.6) were ESBL-PE carriers. Participants with low CD4 count (<350 cells/μL) had significantly higher prevalence of ESBL-PE carriage compared with those with CD4 count ≥350 cells/μL (26/58, 44.8%, vs. 168/537, 31.3%, p = 0.04). Antibiotic use in last 4 weeks (odds ratio [OR] 1.55, 95% CI 1.08–2.22, p = 0.02) and CD4 count ≥350 cells/μL (OR 1.78, 95% CI 1.03–3.09, p = 0.04) were independent risk factors for fecal carriage of ESBL-PE. In total, 244 isolates of ESBL-PE were isolated from 194 participants. Of these, 238/244 (97.5%) harbored blaCTX-M genes, with blaCTX-M-15 being predominant (219/238 (92%), followed by blaCTX-M-27 (9/238 (3.8%), blaCTX-M-14 (8/238 (3.4%), blaCTX-M-55 (1/238), and blaCTX-M 211/3 (1/238). blaSHV-2a genes were detected in four isolates, whereas the blaSHV-12 gene was detected in one isolate. Phenotypic carbapenemase-producing Enterobacteriaceae was detected in one HIV-positive person with CD4 count 132 cells/μL. In conclusion prevalence of ESBL-PE carriage is high among newly diagnosed HIV adults in Dar es Salaam, and is significantly associated antibiotic use and low CD4 count. [ABSTRACT FROM AUTHOR]