Immune surveillance for six vaccinable pathogens using paired plasma and dried blood spots in HIV infected and uninfected children in Kinshasa.

Child vaccination reduces infant mortality rates. HIV-infected children present higher risk of diseases than non-infected. We report the protection coverage rates for 6 vaccine-preventable diseases in a paediatric population from the Democratic Republic of the Congo (DRC) and the impact of HIV infection, providing the first data on the validity of dried blood samples (DBS) to monitor the immune protection. During 2016-2018 DBS from 143 children/adolescents were collected in Kinshasa (DRC), being 52 HIV-infected. Forty-two had a paired plasma sample. Protective IgG was quantified (VirClia-IgG,VIRCELL) to obtain the optimal cut-off in IgG detection in DBS. ROC curves were generated with R software and statistical analyses with Stata. Protective IgG levels varied across pathogens, not reaching herd immunity. HIV-infected presented lower vaccine protection than uninfected for all analyzed pathogens, except rubella, with statistically significant differences for measles (30.8% vs. 53.8%; p = 0.008) and tetanus (3.8% vs. 22%; p = 0.0034). New cut-offs were calculated when using DBS to improve test performance. We reinforce the necessity to increase pediatric vaccination coverage in Kinshasa, especially in HIV seropositive, with less capacity to maintain adequate antibody levels. DBS were useful to monitor vaccination coverage in seroprevalence studies in resource-limited settings, after optimizing the cut-off value for each pathogen.

Simulating the impact of non-pharmaceutical interventions limiting transmission in COVID-19 epidemics using a membrane computing model.

Epidemics caused by microbial organisms are part of the natural phenomena of increasing biological complexity. The heterogeneity and constant variability of hosts, in terms of age, immunological status, family structure, lifestyle, work activities, social and leisure habits, daily division of time and other demographic characteristics make it extremely difficult to predict the evolution of epidemics. Such prediction is, however, critical for implementing intervention measures in due time and with appropriate intensity. General conclusions should be precluded, given that local parameters dominate the flow of local epidemics. Membrane computing models allows us to reproduce the objects (viruses and hosts) and their interactions (stochastic but also with defined probabilities) with an unprecedented level of detail. Our LOIMOS model helps reproduce the demographics and social aspects of a hypothetical town of 10 320 inhabitants in an average European country where COVID-19 is imported from the outside. The above-mentioned characteristics of hosts and their lifestyle are minutely considered. For the data in the Hospital and the ICU we took advantage of the observations at the Nursery Intensive Care Unit of the Consortium University General Hospital, Valencia, Spain (included as author). The dynamics of the epidemics are reproduced and include the effects on viral transmission of innate and acquired immunity at various ages. The model predicts the consequences of delaying the adoption of non-pharmaceutical interventions (between 15 and 45 days after the first reported cases) and the effect of those interventions on infection and mortality rates (reducing transmission by 20, 50 and 80%) in immunological response groups. The lockdown for the elderly population as a single intervention appears to be effective. This modeling exercise exemplifies the application of membrane computing for designing appropriate multilateral interventions in epidemic situations.

Evolutionary Pathways and Trajectories in Antibiotic Resistance.

Evolution is the hallmark of life. Descriptions of the evolution of microorganisms have provided a wealth of information, but knowledge regarding "what happened" has precluded a deeper understanding of "how" evolution has proceeded, as in the case of antimicrobial resistance. The difficulty in answering the "how" question lies in the multihierarchical dimensions of evolutionary processes, nested in complex networks, encompassing all units of selection, from genes to communities and ecosystems. At the simplest ontological level (as resistance genes), evolution proceeds by random (mutation and drift) and directional (natural selection) processes; however, sequential pathways of adaptive variation can occasionally be observed, and under fixed circumstances (particular fitness landscapes), evolution is predictable. At the highest level (such as that of plasmids, clones, species, microbiotas), the systems' degrees of freedom increase dramatically, related to the variable dispersal, fragmentation, relatedness, or coalescence of bacterial populations, depending on heterogeneous and changing niches and selective gradients in complex environments. Evolutionary trajectories of antibiotic resistance find their way in these changing landscapes subjected to random variations, becoming highly entropic and therefore unpredictable. However, experimental, phylogenetic, and ecogenetic analyses reveal preferential frequented paths (highways) where antibiotic resistance flows and propagates, allowing some understanding of evolutionary dynamics, modeling and designing interventions. Studies on antibiotic resistance have an applied aspect in improving individual health, One Health, and Global Health, as well as an academic value for understanding evolution. Most importantly, they have a heuristic significance as a model to reduce the negative influence of anthropogenic effects on the environment.

Acute-onset smell and taste disorders in the context of COVID-19: a pilot multicentre polymerase chain reaction based case-control study.

BACKGROUND AND PURPOSE: Specific respiratory tract infections, including COVID-19, may cause smell and/or taste disorders (STDs) with increased frequency. The aim was to determine whether new-onset STDs are more frequent amongst COVID-19 patients than influenza patients. METHOD: This was a case-control study including hospitalized patients of two tertiary care centres. Consecutive patients positive for COVID-19 polymerase chain reaction (cases) and patients positive for influenza polymerase chain reaction (historical control sample) were assessed during specific periods, employing a self-reported STD questionnaire. RESULTS: Seventy-nine cases and 40 controls were included. No significant differences were found in basal features between the two groups. New-onset STDs were significantly more frequent amongst cases (31, 39.2%) than in the control group (5, 12.5 %) [adjusted odds ratio 21.4 (2.77-165.4, P = 0.003)]. COVID-19 patients with new-onset STDs were significantly younger than COVID-19 patients without STDs (52.6 ± 17.2 vs. 67.4 ± 15.1, P < 0.001). Amongst COVID-19 patients who presented STDs, 22 (70.9%) recalled an acute onset and it was an initial manifestation in 11 (35.5%). Twenty-five (80.6%) presented smell disorders (mostly anosmia, 14, 45.2%) and 28 (90.3%) taste disorders (mostly ageusia, 14, 45.2%). Only four (12.9 %) reported concomitant nasal obstruction. The mean duration of STD was 7.5 ± 3.2 days and 12 patients (40%) manifested complete recovery after 7.4 ± 2.3 days of onset. CONCLUSION: New-onset STDs were significantly more frequent amongst COVID-19 patients than influenza patients; they usually had an acute onset and were commonly an initial manifestation. The use of STD assessment in anamnesis as a hint for COVID-19 and to support individuals' self-isolation in the current epidemic context is suggested.

Spread of a new Chlamydia trachomatis variant from men who have sex with men to the heterosexual population after replacement and recombination in ompA and pmpH genes.

OBJECTIVES: Sexually transmitted infections are frequently related to outbreaks in high-risk populations due to the dense sexual networks. We wanted to determine the dissemination of a Chlamydia trachomatis variant characterized by the pmpH-recombinant gene between L and G genotypes, which was previously described in a high-risk population. METHODS: A total of 449 samples were analysed in two periods ranging from 2009 to 2015 for detection of the pmpH-recombinant gene. For those samples yielding positive amplification, a sampling was selected for phylogenetic reconstructions based on sequencing of five chromosomal genes. RESULTS: Globally this variant was found in 113 of the 449 samples (25%). During the first years (2009-13), this variant was found almost exclusively in rectal samples (30/112 samples) of men who have sex with men and in only one non-rectal sample (1/63). In 2014, this variant was also found in urethral and pharyngeal samples (1/24 and 1/7, respectively). However, in 2015, an epidemiological change was observed as the proportion of this variant had increased in rectal samples (20/51; 39%) and non-rectal samples, including cervical samples (51/142; 36.4%). The molecular characterization revealed the replacement of the ompA gene belonging to subtype G in samples recovered from 2009 to 2013 by the ompA gene belonging to subtype J after 2013. CONCLUSIONS: Our data would support the evidence that subtype J could be a 'subtype bridge' between different sexual networks, as subtype J has been found in men who have sex with men and heterosexual populations in similar proportions. This work reveals the necessity of implementing molecular surveillance in extra-rectal samples to help us understand the gaps in transmission.

Clinical and epidemiological characterization of a lymphogranuloma venereum outbreak in Madrid, Spain: co-circulation of two variants.

The lymphogranuloma venereum (LGV) outbreak described in the Netherlands in 2003, increased the interest in the genotyping of Chlamydia trachomatis. Although international surveillance programmes were implemented, these studies slowly decreased in the following years. Now data have revealed a new accumulation of LGV cases in those European countries with extended surveillance programmes. Between March 2009 and November 2011, a study was carried out to detect LGV cases in Madrid. The study was based on screening of C. trachomatis using commercial kits, followed by real-time pmpH-PCR discriminating LGV strains, and finally ompA gene was sequenced for phylogenetic reconstruction. Ninety-four LGV infections were identified. The number of cases increased from 10 to 30 and then to 54 during 2009-2011. Incidence of LGV was strongly associated with men who have sex with men; but in 2011, LGV cases were described in women and heterosexual men. Sixty-nine patients were also human immunodeficiency virus (HIV) positive, with detectable viral loads at the moment of LGV diagnosis, suggesting a high-risk of co-transmission. In fact, in four patients the diagnosis of HIV was simultaneous with LGV infection. The conventional treatment with doxycycline was prescribed in 75 patients, although in three patients the treatment failed. The sequencing of the ompA gene permitted identification of two independent transmission nodes. One constituted by 25 sequences identical to the L2b variant, and a second node including 37 sequences identical to L2. This epidemiological situation characterized by the co-circulation of two LGV variants has not been previously described, reinforcing the need for screening and genotyping of LGV strains.

Effect of food on the antiviral activity of didanosine enteric-coated capsules: a pilot comparative study.

OBJECTIVES: To determine the effect of food on the antiviral activity of enteric-coated (EC) capsules of didanosine (ddI). METHODS: We conducted a pilot, randomized, open-label study of 28-day ddI-EC capsules monotherapy-administered in a fasted state (group 1, n=11) or with food (group 2, n=10) to treatment-naïve chronically HIV-1-infected individuals. To assess the antiviral efficacy, HIV-1 RNA was determined at baseline, day 3, day 7 and weekly thereafter. The area under the HIV-1 RNA curve minus baseline weighted by time (AUCMB/day) was calculated. RESULTS: Mean baseline HIV-1 RNA was 4.2 log(10) copies/mL in group 1 and 3.8 log(10) copies/mL in group 2. After 28 days, the mean HIV-1 RNA reduction was 0.99 log(10) copies/mL [95% confidence interval (CI) 0.45-1.53] for group 1 and 0.89 log(10) copies/mL (95% CI 0.38-1.40) for group 2. AUCMB/day values were 0.775 log(10) copies/mL (95% CI 0.33-1.22) and 0.774 log(10) copies/mL (95% CI 0.48-1.07), respectively, showing no difference in the rate of decrease of HIV-1 RNA (P=0.995). Mean ddI plasma levels at day 28 were 0.0234 mg/L for group 1 and 0.0227 mg/L for group 2 (P=0.96). CONCLUSIONS: In this pilot study, the administration of food did not have any significant effect on the antiviral activity of ddI-EC capsules.

[Trans-gram gene transfer: the case of beta-lactamases]. / Transferencia génica trans-gram: el caso de las betalactamasas.

Lateral or horizontal gene transfer is a phenomenon that has influenced the evolution of microorganisms. Despite the evolutionary trend toward genetic isolation, lateral transfer seems to take place relatively frequently, bridging the gap between very separate species. The acquisition of foreign genes may have accelerated in recent years because of the increase in the adaptive needs of bacteria, particularly through the use of antibiotics. Transfer of genes encoding beta-lactamases from Gram-positive to Gram-negative bacteria (trans-gram transfer) may be suggested on the basis of sequence analysis. We found that the sequences of the beta-lactamases BRO-1 and ACl-1, from the Gram-negative bacterial organisms Moraxella and Acidaminococcus, respectively are abnormally placed among sequences from Gram-positive beta-lactamases in phylogenetic trees. In both cases, the topology of the enzyme (attached to the cellular membrane), the structure of the signal peptide, and the Shine-Dalgarno region suggest that these enzymes originated from Gram-positive organisms. Results remain inconclusive for Haemophilus ROB-1 beta-lactamase.

In vitro activity of telithromycin against Spanish Streptococcus pneumoniae isolates with characterized macrolide resistance mechanisms.

The susceptibilities to telithromycin of 203 Streptococcus pneumoniae isolates prospectively collected during 1999 and 2000 from 14 different geographical areas in Spain were tested and compared with those to erythromycin A, clindamycin, quinupristin-dalfopristin, penicillin G, cefotaxime, and levofloxacin. Telithromycin was active against 98.9% of isolates (MICs, < or =0.5 microg/ml), with MICs at which 90% of isolates are inhibited being 0.06 microg/ml, irrespective of the resistance genotype. The corresponding values for erythromycin were 61.0% (MICs, < or =0.25 microg/ml) and >64 microg/ml. The erm(B) gene (macrolide-lincosamide-streptogramin B resistance phenotype) was detected in 36.4% (n = 74) of the isolates, which corresponded to 93.6% of erythromycin-intermediate and -resistant isolates, whereas the mef(A) gene (M phenotype [resistance to erythromycin and susceptibility to clindamycin and spiramycin without blunting]) was present in only 2.4% (n = 5) of the isolates. One of the latter isolates also carried erm(B). Interestingly, in one isolate for which the erythromycin MIC was 2 microg/ml, none of these resistance genes could be detected. Erythromycin MICs for S. pneumoniae erm(B)-positive isolates were higher (range, 0.5 to >64 microg/ml) than those for erm(B)- and mef(A)-negative isolates (range, 0.008 to 2 microg/ml). The corresponding values for telithromycin were lower for both groups, with ranges of 0.004 to 1 and 0.002 to 0.06 microg/ml, respectively. The erythromycin MIC was high for a large number of erm(B)-positive isolates, but the telithromycin MIC was low for these isolates. These results indicate the potential usefulness of telithromycin for the treatment of infections caused by erythromycin-susceptible and -resistant S. pneumoniae isolates when macrolides are indicated.

ACI-1 from Acidaminococcus fermentans: characterization of the first beta-lactamase in Anaerobic cocci.

Acidaminococcus fermentans belongs to the group of strictly anaerobic gram-negative cocci. All previously described Acidaminococcus strains are susceptible to beta-lactam antibiotics. An A. fermentans strain (RYC-MR95) resistant to penicillin and expanded-spectrum cephalosporin (amoxicillin and cefotaxime MICs, 64 microgram/ml) was isolated from a human perianal abscess. A fragment encoding a beta-lactamase from genomic DNA was cloned in Escherichia coli K-12 strain HB101, and the recombinant strain expressed resistance to amoxicillin (MIC, 1,024 microgram/ml) and cefotaxime (MIC, 4 microgram/ml). Clavulanic acid decreased the MICs to 8 and 0.03 microgram/ml, respectively. Analysis of the nucleotide sequence revealed a new class A beta-lactamase, ACI-1. In accordance with its biochemical properties, we propose to assign ACI-1 to functional group 2be. The ACI-1 enzyme (estimated pI 4.3) had <50% amino acid identity with any other class A beta-lactamases, the closest being ROB-1 from Haemophilus influenzae (44%). ACI-1 was closer to class A beta-lactamases from some gram-positive organisms (41 to 44% amino acid identity with Bacillus beta-lactamases) than to most class A enzymes from gram-negative organisms (TEM-1, 24.6%). The aci1 gene had a G+C content of 42.1%, in contrast with 56% G+C content for genomic DNA from A. fermentans, thus suggesting that aci1 may have been obtained by horizontal gene transfer.

Mycobacterium tuberculosis subsp. caprae subsp. nov.: a taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain.

Isolates from the Mycobacterium tuberculosis complex cultured from caprine pathological tissue samples were biochemically and genetically characterized. The isolates were negative for nitrate reduction and niacin accumulation, they weakly hydrolysed Tween 80, were sensitive to pyrazinamide (50 micrograms ml-1) and were resistant to 1 and 2 micrograms tiophene-2-carboxylic acid hydrazide ml-1 but not to 5 or 10 micrograms tiophene-2-carboxylic acid hydrazide ml-1. Sequencing of the pncA gene revealed a polymorphism characteristic of M. tuberculosis, whereas oxyR, katG and gyrA sequences were characteristic of Mycobacterium bovis. The fingerprinting patterns obtained with IS6110, direct repeats and polymorphic G+C-rich sequence-associated RFLP and direct variable repeat-spacer oligonucelotide typing (spoligotyping) segregated these isolates from the other members of the complex. The results of this testing, together with the repeated association of this micro-organism with goats, suggest that a new member of this taxonomic complex not matching any of the classical species had been identified. This unusual mycobacterium may play a role in the epidemiology of animal and human tuberculosis in Spain. The name Mycobacterium tuberculosis subsp. caprae subsp. nov. is proposed for these isolates. The type strain of Mycobacterium tuberculosis subsp. caprae subsp. nov. is gM-1T (= CIP 105776T).

Molecular markers demonstrate that the first described multidrug-resistant Mycobacterium bovis outbreak was due to Mycobacterium tuberculosis.

We genetically characterized multidrug-resistant Mycobacterium tuberculosis complex strains which caused a nosocomial outbreak of tuberculosis affecting six human immunodeficiency virus (HIV)-positive patients and one HIV-negative staff member (E. Bouvet, E. Casalino, G. Mendoza-Sassi, S. Lariven, E. Vallée, M. Pernet, S. Gottot, and F. Vachon, AIDS 7:1453-1460, 1993). The strains showed all the phenotypic characteristics of Mycobacterium bovis. They presented a high copy number of IS6110, the spacers 40 to 43 in the direct repeat locus, and the mtp40 fragment. They lacked the G-A mutation at position 285 in the oxyR gene and the C-G mutation at position 169 in the pncA gene. These genetic characteristics revealed that these were dysgonic, slow-growing M. tuberculosis strains mimicking the M. bovis phenotype, probably as a consequence of cellular alterations associated with the multidrug resistance. Spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) analysis confirmed that the outbreak was due to a single strain. However, the IS6110 RFLP pattern of the strain isolated from the last patient, diagnosed three years after the index case, differed slightly from the patterns of the other six strains. A model of a possible genetic event is presented to explain this divergence. This study stresses the value of using several independent molecular markers to identify multidrug-resistant tubercle bacilli.

Allele-specific PCR method based on pncA and oxyR sequences for distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: intraspecific M. bovis pncA sequence polymorphism.

An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform.