Pedro L. Alonso

Intracontinental spread of human invasive Salmonella Typhimurium pathovariants in sub-Saharan Africa

A highly invasive form of non-typhoidal Salmonella (iNTS) disease has recently been documented in many countries in sub-Saharan Africa. The most common Salmonella enterica serovar causing this disease is Typhimurium (Salmonella Typhimurium). We applied whole-genome sequence–based phylogenetic methods to define the population structure of sub-Saharan African invasive Salmonella Typhimurium isolates and compared these to global Salmonella Typhimurium populations. Notably, the vast majority of sub-Saharan invasive Salmonella Typhimurium isolates fell within two closely related, highly clustered phylogenetic lineages that we estimate emerged independently ∼52 and ∼35 years ago in close temporal association with the current HIV pandemic. Clonal replacement of isolates from lineage I by those from lineage II was potentially influenced by the use of chloramphenicol for the treatment of iNTS disease. Our analysis suggests that iNTS disease is in part an epidemic in sub-Saharan Africa caused by highly related Salmonella Typhimurium lineages that may have occupied new niches associated with a compromised human population and antibiotic treatment.

Plasmodium falciparum multiple infections in Mozambique, its relation to other malariological indices and to prospective risk of malaria morbidity.

We describe the frequency of Plasmodium falciparum clones infecting individuals living in a rural area of southern Mozambique and analyse the relationship between multiplicity of infection, age and other malariometric indices, including prospective risk of clinical malaria. The genotyping was based on the use of restriction fragment length polymorphism–polymerase chain reaction (RFLP–PCR) analysis of P. falciparum merozoite surface protein 2 (msp2). We analysed 826 samples collected during five cross‐sectional surveys from residents of Manhiça ranging in age from 4 months to 83 years. We also determined the multiplicity of infection in samples obtained from 6‐month‐old infants (n = 79) and children <10 years (n = 158) who were then treated and followed prospectively for 1 year or 75 weeks, respectively. Multiplicity of infection did not vary significantly during the first year of life, but increased thereafter, and decreased during adulthood to the levels found in infants. With increasing multiplicity of infection, there was a statistically significant decrease in the risk of submicroscopic infections. There was also a significant correlation between multiplicity of infection and parasite density in infants, children <4 years of age and adults, suggesting that high densities increase the probability of discriminating more clones in complex infections. We found that the relationship between multiple infections and malaria morbidity is age‐dependent. In infants, the risk of subsequent episodes of clinical malaria was related to the parasite density but not to baseline multiplicity of infection. In older children, however, the more clones a child carried, the more likely they were to have a clinical malaria episode, and this was true after adjusting for parasite densities. This change in the association between multiplicity and risk of clinical malaria may indicate a shift in the host response to P. falciparum.

Vaccine-related HPV genotypes in women with and without cervical cancer in Mozambique: Burden and potential for prevention

Knowledge about the burden of Human Papillomavirus (HPV) infections in Sub‐Saharan Africa is very limited. We collected cervical samples from 262 women from the general population and 241 tumor samples from women with invasive cervical cancer in Mozambique and tested them for HPV genotyping by the SPF10‐LiPA25 PCR system. Among the 195 women without cervical abnormalities by cytology HPV prevalence was 75.9%. In this group of women, the most frequently identified HPV types among HPV‐positive women were in descending order of frequency: HPV51 (23.6%), HPV35 (19.6%), HPV18 (14.2%), HPV31 (13.5%) and HPV52 (12.8%). In women with cervical cancer HPV DNA detection was 100%. The type‐specific distribution of the most frequent types in descending order of frequency was: HPV16 (47.0%), HPV18 (31.3%), HPV51 (14.8%), HPV52 (14.3%), HPV45 (12.6%), HPV35 (10.4%), HPV33 (4.8%) and HPV31 (2.6%). HPVs 16/18 and HPVs 16/18/31/45 were detected in 71.7% and 80.9% of cervical cancer tissue, respectively. While HPVs 51 and 35 were the two most common types in cytologically normal women in Mozambique, HPVs 16 and 18 remained the two most frequently identified types in cervical cancer. The introduction of an efficacious HPV 16/18 vaccine could potentially prevent the occurrence of 72% of cervical cancer cases and up to 81% of the cases if full cross‐protection against HPVs 31 and 45 is assumed.

Severe malnutrition among children under the age of 5 years admitted to a rural district hospital in southern Mozambique.

OBJECTIVE To describe the burden, clinical characteristics and prognostic factors of severe malnutrition in children under the age of 5 years. DESIGN Retrospective study of hospital-based data systematically collected from January 2001 to December 2010. SETTING Rural Mozambican district hospital. SUBJECTS All children aged <5 years admitted with severe malnutrition. RESULTS During the 10-year long study surveillance, 274 813 children belonging to Manhiça’s Demographic Surveillance System were seen at out-patient clinics, almost half of whom (47 %) presented with some indication of malnutrition and 6% (17 188/274 813) with severe malnutrition. Of these, only 15% (2522/17 188) were eventually admitted. Case fatality rate of severe malnutrition was 7% (162/2274). Bacteraemia, hypoglycaemia, oral candidiasis, prostration, oedema, pallor and acute diarrhoea were independently associated with an increased risk of in-hospital mortality, while malaria parasitaemia and breast-feeding were independently associated with a lower risk of a poor outcome. Overall minimum communitybased incidence rate was 15 cases per 1000 child-years at risk and children aged 12–23 months had the highest incidence. CONCLUSIONS Severe malnutrition among admitted children in this Mozambican setting was common but frequently went undetected, despite being associated with a high risk of death. Measures to improve its recognition by clinicians responsible for the first evaluation of patients at the out-patient level are urgently needed so as to improve their likelihood of survival. Together with this, the rapid management of complications such as hypoglycaemia and concomitant co-infections such as bacteraemia, acute diarrhoea, oral candidiasis and HIV/AIDS may contribute to reverse the intolerable toll that malnutrition poses in the health of children in rural African settings.

Low paediatric tuberculosis case detection rate in Southern Mozambique.

Two core indicators adopted for evaluating tuberculosis (TB) control programmes are treatment outcome and case detection rate (CDR). While the former is easy to report, the CDR can only be estimated (calculated as notifications of new and relapse cases divided by estimated incidence). According to the World Health Organization (WHO), Mozambique has one of the lowest CDRs among the high TB burden countries (HBCs), with 37% in 2013 [1]. In children, calculating CDR is even more challenging, given the difficulty in diagnosing TB and the lack of accurate estimates for paediatric incidence [2]. Several paediatric TB incidence estimates have been published recently [3–5], showing higher figures than those provided by WHO. The large variation in estimates and the lack of population-based data from HBCs (particularly from Sub-Saharan Africa) highlights the urgent need for new data to inform predictive models necessary to implement the “End TB Strategy” and achieve elimination [6]. The objective of this study was to calculate the paediatric CDR in Mozambique and to provide reference methodology and evidence for other countries. In this study, the case detection rate of TB in children <3 years in Manhiça, Mozambique was estimated to be 40.8% http://ow.ly/UQg0g

Recent HIV-1 infection: identification of individuals with high viral load setpoint in a voluntary counselling and testing centre in rural Mozambique.

Background Identification of recent HIV-infections is important for describing the HIV epidemic and compiling HIV-RNA-setpoint data for future HIV intervention trials. We conducted a study to characterize recent infections, and HIV-RNA-setpoint within the adult population presenting at a voluntary counselling and testing centre (VCT) in southern Mozambique. Methods All adults attending the Manhiça District-Hospital VCT between April and October 2009 were recruited if they had at least one positive rapid HIV-serology test. Patients were screened for recent HIV-1 infection by BED-CEIA HIV-incidence test. Clinical examination, assessment of HIV-RNA and CD4 cell counts were performed at enrollment, 4 and 10 months. Results Of the 492 participants included in this study, the prevalence of recent infections as defined by BED-CEIA test, CD4 counts >200 cells/µl and HIV-RNA >400 copies/mL, was 11.58% (57/492; 95% CI 8.89–14.74). Due to heterogeneity in HIV-RNA levels in recently infected patients, individuals were categorized as having “high” HIV-RNA load if their HIV-RNA level was above the median (4.98 log10 copies/mL) at diagnosis. The “high” HIV-RNA group sustained a significantly higher HIV-viral load at all visits with a median HIV-RNA setpoint of 5.22 log10 copies/mL (IQR 5.18–5.47) as compared to the median of 4.15 log10 copies/ml (IQR 3.37–4.43) for the other patients (p = 0.0001). Conclusion The low proportion of recent HIV-infections among HIV-seropositive VCT clients suggests that most of this population attends the VCT at later stages of HIV/AIDS. Characterization of HIV-RNA-setpoint may serve to identify recently infected individuals maintaining HIV viral load>5 log10 copies/mL as candidates for antiretroviral treatment as prevention interventions.

A variant in the gene FUT9 is associated with susceptibility to placental malaria infection

Malaria in pregnancy forms a substantial part of the worldwide burden of malaria, with an estimated annual death toll of up to 200 000 infants, as well as increased maternal morbidity and mortality. Studies of genetic susceptibility to malaria have so far focused on infant malaria, with only a few studies investigating the genetic basis of placental malaria, focusing only on a limited number of candidate genes. The aim of this study therefore was to identify novel host genetic factors involved in placental malaria infection. To this end we carried out a nested case-control study on 180 Mozambican pregnant women with placental malaria infection, and 180 controls within an intervention trial of malaria prevention. We genotyped 880 SNPs in a set of 64 functionally related genes involved in glycosylation and innate immunity. A single nucleotide polymorphism (SNP) located in the gene FUT9, rs3811070, was significantly associated with placental malaria infection (odds ratio = 2.31, permutation P-value=0.028). Haplotypic analysis revealed a similarly strong association of a common haplotype of four SNPs including rs3811070. FUT9 codes for a fucosyl-transferase that is catalyzing the last step in the biosynthesis of the Lewis-x antigen, which forms part of the Lewis blood group-related antigens. These results therefore suggest an involvement of this antigen in the pathogenesis of placental malaria infection.

Molecular Characterization of Community Acquired Staphylococcus aureus Bacteremia in Young Children in Southern Mozambique, 2001–2009

Background: The emergence of community-acquired Staphylococcus aureus infections is increasingly recognized as life threating problem worldwide. In Manhiça district, southern Mozambique, S. aureus is the leading cause of community-acquired bacteremia in neonates. Methods: Eighty-four S. aureus isolates from children less than 5 years admitted to Manhiça District Hospital from 2001 to 2009 were randomly selected and genetically characterized by DNA microarray and spa typing. Antimicrobial susceptibility was determined by VITEK 2. Results: Thirty-eight different spa types and 14 clonal complexes (CC) were identified. Spa-type t084 (n = 10; 12%) was the most predominant while CC8 (n = 18; 21%) and CC15 (n = 14; 16%) were the most frequent CCs. Mortality tended to be higher among children infected with CC45 (33.3%, 1/3) and CC8 (27.8%, 5/18). The majority of isolates possessed the accessory gene regulator I (45%) and belonged to either capsule type 8 (52%) or 5 (47%). Panton valentine leukocidin (PVL) encoding genes were detected in 30%. Antibiotic resistance was high for penicillin (89%), tetracycline (59%) and Trimethoprim Sulfamethoxazole (36%) while MRSA was uncommon (8%). Conclusions: Although MRSA were uncommon, we found high genetic diversity of methicillin susceptible S. aureus causing bacteremia in Mozambican children, associated with high resistance to the most available antibiotics in this community. Some CCs are likely to be more lethal indicating the need for prompt recognition and appropriate treatment.

Characterisation of extended-spectrum b-lactamases among Klebsiella pneumoniae isolates causing bacteraemia and urinary tract infection in Mozambique

The aim of this study was to determine the prevalence of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolated from urinary tract and bloodstream infections in a rural hospital in Manhiça, Mozambique. ESBLs were investigated among ceftriaxone-non-susceptible K. pneumoniae clinical isolates recovered between 2004 and 2009. Characterisation of blaCTX-M, blaSHV, blaOXA and blaTEM genes was performed by PCR and sequencing. Epidemiological relationships were established by phylogenetic analysis, repetitive extragenic palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), whilst plasmid transferability was evaluated by conjugation. In addition, the presence of class 1 and 2 integrons was studied. A total of 19 K. pneumoniae were analysed. The blaCTX-M-15 gene was found in all strains. Other ESBL genes were found concomitantly, including blaSHV-5, blaSHV-2, blaSHV-2A, blaSHV-12 and blaSHV-38. In addition, other β-lactamases such as blaTEM-1 and blaOXA-30 were also detected. REP-PCR identified 15 different epidemiological profiles. MLST analysis also showed great variability of sequence types. The blaCTX-M-15 gene showed a high transfer capacity. The presence of class 1 integrons was high. High levels of multidrug resistance were also found. In conclusion, these data show the dominance of the CTX-M-type ESBL, particularly CTX-M-15, supporting its worldwide dissemination, including in areas with limited access to third-generation cephalosporins. This finding is a matter of concern for clinical management as third-generation cephalosporins are an alternative for treating severe cases of multidrug-resistant infections in this community.

Vacunas contra la malaria, una prometedora espera

Los prometedores resultados ofrecidos por la vacuna RTS,S/AS02A en ensayos clinicos indican que el desarrollo de una vacuna eficaz es posible. Puntos clave El principio del siglo XXI esta siendo testigo de un renovado afan cientifico por desarrollar una vacuna eficaz contra la malaria. Los enormes progresos que se han producido en los ultimos anos, junto con los primeros exitos cosechados en ensayos clinicos de vacunas candidatas, permiten un razonable optimismo acerca del desarrollo en un futuro proximo de una vacuna. Aunque posiblemente no se consiga prevenir completamente la infeccion, las nuevas vacunas seran capaces de disminuir las consecuencias de una enfermedad responsable de una enorme morbi-mortalidad, predominantemente pediatrica. En este articulo se revisan los principales conceptos teoricos relacionados con el desarrollo de vacunas de malaria.