Implementation of multidrug-resistant tuberculosis (MDR-TB) treatment in Gabon: lessons learnt from the field.

PURPOSE: Since May 2016, WHO recommended a 9-12 month short-treatment regimen for multidrug-resistant tuberculosis (MDR-TB) treatment known as the 'Bangladesh Regimen'. However, limited data exist on the appropriateness thereof, and its implementation in low- and middle-income countries (LMIC). We report here on the pilot phase of the evaluation of the Bangladesh regimen in Gabon, prior to its endorsement by the WHO. METHODS: This ongoing observational study started in September 2015. Intensive training of hospital health workers as well as community information and education were conducted. GeneXpert-confirmed MDR-TB patients received the second-line anti-tuberculosis drugs (4KmMfxPtoHCfzEZ/5MfxCfzEZ). Sputum smears and cultures were done monthly. Adverse events were monitored daily. RESULTS: Eleven patients have been treated for MDR-TB piloting the short regimen. All were HIV-negative and presented in poor health with extensive pulmonary lesions. The overall sputum culture conversion rate was 64% after 4 months of treatment. Three patients developed marked hearing loss; one a transient cutaneous rash. Of 11 patients in our continuous care, 7 (63.6%) significantly improved clinically and bacteriologically. One (9.1%) patient experienced a treatment failure, two (18.2%) died, and one (9.1%) was lost to follow up. CONCLUSIONS: Our pioneering data on systematic MDR-TB treatment in Gabon, with currently almost total absence of resistance against the second-line drugs, demonstrate that a 9-month regimen has the capacity to facilitate early culture negativity and sustained clinical improvement. Close adverse events monitoring and continuous care are vital to success.

Demography, maternal health and the epidemiology of malaria and other major infectious diseases in the rural department Tsamba-Magotsi, Ngounie Province, in central African Gabon.

BACKGROUND: Sub-Saharan Africa is undergoing an epidemiological transition from a predominance of infectious diseases to non-communicable and lifestyle related conditions. However, the pace of this transition and the pattern of disease epidemiology are uneven between affluent urban and rural poor populations. To address this question for a remote rural region located in the central African rainforest region of Gabon, this study was conducted to assess reasons for health care attendance and to characterize the epidemiology of malaria and other major infectious diseases for the department of Tsamba Magotsi. METHODS: Major causes for health care attendance were collected from local hospital records. Cross sectional population based surveys were performed for the assessment of local malaria epidemiology. Pregnant women attending antenatal care services were surveyed as a sentinel population for the characterization of chronic viral and parasitic infections in the community. RESULTS: Infectious diseases were responsible for 71% (7469) of a total of 10,580 consultations at the formal health care sector in 2010. Overall, malaria - defined by clinical syndrome - remained the most frequent cause for health care attendance. A cross sectional malaria survey in 840 asymptomatic individuals residing in Tsamba Magotsi resulted in a Plasmodium spp. infection prevalence of 37%. The infection rate in 2-10 year old asymptomatic children - a standard measure for malaria endemicity - was 46% (100 of 217) with P. falciparum as predominant species (79%). Infection with other plasmodial species (P. ovale and P. malariae) presented most commonly as coinfections (23.2%). Prevalence of HIV, HBV, and syphilis were 6.2, 7.3, and 2.5%, respectively, in cross-sectional assessments of antenatal care visits of pregnant women. Urogenital schistosomiasis and the filarial pathogens Loa loa and Mansonella perstans are highly prevalent chronic parasitic infections affecting the local population. CONCLUSIONS: Despite major improvements in the accessibility of Tsamba Magotsi over the past decade the epidemiological transition does not appear to have majorly changed on the spectrum of diseases in this rural Gabonese population. The high prevalence of Plasmodium infection indicates a high burden of malaria related morbidity. Infectious diseases remain one of the most important health issues and further research activities in the field of tropical medicine and infectious diseases could help improve health care for the local population.

Genetic Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine.

BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Carriage of encapsulated bacteria in Gabonese children with sickle cell anaemia.

Sickle cell anaemia (SCA) is a haemoglobin disorder that alters the deformability of erythrocytes through abnormal polymerization of haemoglobin. Children with SCA have an increased risk of infections with encapsulated bacteria. To guide the antibiotic prophylaxis and vaccinations in children with SCA in Gabon, we characterized Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae from children with and without SCA. We performed a cross-sectional study and compared nasal and pharyngeal S. pneumoniae, Staph. aureus and H. influenzae isolates from SCA children (n = 73) with comparators matched for age, residence and sex (n = 143) in a matched-comparison analysis. The resistance pattern and capsular type were identified for each isolate. The total carriage rate for S. pneumoniae, Staph. aureus and H. influenzae was 13.8%, 46.7% and 12.5%, respectively, and did not differ between groups (p >0.05). The mean number of days under antibiotic treatment in the past year was higher in children with SCA than in controls (penicillin: 70.1 vs 0.1 days, p 0.00002). The total non-susceptibility rate was 30% for oral and parenteral (meningitis) penicillin in S. pneumoniae, resistance rates were 1.6% for oxacillin in Staph. aureus and 14.8% for ampicillin in H. influenzae. Susceptibility to antibiotic agents and distribution of capsular types did not differ significantly between both groups. In conclusion, carriage and resistance rates are similar in children with and without SCA. Our data provide the basis to guide empiric therapy of invasive diseases caused by S. pneumoniae, Staph. aureus and H. influenza in children in Gabon.

Clonal structure of Staphylococcus aureus colonizing children with sickle cell anaemia and healthy controls.

Children with sickle cell anaemia (SCA) might carry hospital-associated bacterial lineages due to frequent hospital stays and antibiotic treatments. In this study we compared Staphylococcus aureus from SCA patients (n=73) and healthy children (n=143) in a cross-sectional study in Gabon. S. aureus carriage did not differ between children with SCA (n=34, 46∙6%) and controls matched for age, residence and sex (n=67, 46∙9%). Both groups shared similar S. aureus genotypes. This finding points towards a transmission of S. aureus between both groups in the community. We conclude that resistance rates from population-based studies with healthy participants could therefore also be used to guide treatment and prophylaxis of endogenous infections in children with SCA despite a different selection pressure.

Virulence factors and genotypes of Staphylococcus aureus from infection and carriage in Gabon.

Staphylococcus aureus isolates from developed countries have been extensively analyzed with respect to their virulence patterns and clonal relatedness but there is only sparse information on the molecular diversity of S. aureus isolates from Africa. In particular, little is known about S. aureus isolates from asymptomatic carriers compared with isolates causing infections. From 2008 to 2010, we prospectively collected S. aureus isolates from asymptomatic carriers and infections in Lambaréné, Gabon, Central Africa. For these isolates, we determined major virulence factors, and performed multilocus sequence typing (MLST) and spa typing. Among 163 S. aureus isolates from asymptomatic carriers, we found the MLST clonal complexes (CCs) 5, 6, 7, 8, 9, 15, 25, 30, 45, 88, 101, 121 and 152; 3.7% were methicillin-resistant (MRSA). The clinical isolates were associated with CCs 5, 8, 9, 15, 88, 121 and 152; 11% were MRSA. Sequence types 1 and 88 were significantly associated with infection and sequence type 508 was associated with carriage. Remarkably, there was a high prevalence of Panton-Valentine leukocidin (PVL) -encoding genes both in disease-related isolates (57.4%) and in carrier isolates (40.5%). We found differences in the clonal structure and virulence pattern of Gabonese S. aureus isolates from asymptomatic carriers and infections. Of note, S. aureus isolates from Gabon show a very high prevalence of PVL-encoding genes, which exceeds the rates observed for developed countries.

Cellular and humoral responses to tetanus vaccination in Gabonese children.

Protection to tetanus is often not optimal in developing countries due to incomplete vaccination schemes, or decreased efficacy of vaccination. In this study we investigated the immunological response to tetanus booster vaccination in school children living in a semi-urban or in a rural area of Gabon. Tetanus-specific total IgG as well as antibody subclasses of the IgG1, IgG2, IgG3 and IgG4 isotype and the avidity of the dominating IgG1 subclass were determined both before and 1 month after the booster vaccination. In addition, tetanus-specific cytokine responses were determined. We found a polarization towards a T helper 1 (Th1) profile in the semi-urban children, whereas the cytokine responses of the rural children showed a T helper 2 (Th2) skewed response. Furthermore, tetanus-specific antibodies of the different IgG subclasses were all increased upon a tetanus booster vaccination and levels of IgG1 and IgG3 were higher in the rural children. In conclusion, a tetanus booster vaccination induced a stronger Th2 over Th1 cytokine profile to tetanus toxoid (TT) in rural children who showed the highest levels of IgG1 and IgG3 anti-TT antibody responses.

Cellular and humoral responses to influenza in gabonese children living in rural and semi-urban areas.

BACKGROUND: With the current attention to the pandemic threat of avian influenza viruses, it is recognized that there is little information on influenza in Africa. In addition, the effects of influenza vaccination in African countries could be very different from the effects in regions with less exposure to microorganisms and parasites. METHODS: To monitor the presence of influenza viruses and investigate the immunological responses to influenza vaccination, schoolchildren in semi-urban and rural regions of Gabon were studied. Influenza-specific antibody responses to the 3 strains represented in the vaccine were determined in the serum. Furthermore, cytokine responses were measured after in vitro stimulation of whole blood by influenza antigens, before and after vaccination. RESULTS: Prevaccination titers of antibody against H3N2 were high. At vaccination, the titers of antibody against the 3 influenza strains increased significantly. The anti-H1N1 and anti-B responses after vaccination were weaker in rural schoolchildren than in semi-urban schoolchildren. Influenza-specific cytokine responses were induced within a week, showing significantly lower interferon- gamma and significantly higher interleukin-5 in the children from rural areas. CONCLUSIONS: Prevaccination antibody levels indicated that influenza viruses circulate in Gabon. Altogether, influenza vaccination induces weaker immune responses in a rural population than in a semi-urban population of Gabonese schoolchildren.

Ganglioside (GD2) expression and intermediary filaments in astrocytic tumors.

The search of proliferation markers in astrocytic tumors that may serve as targets for therapeutic interventions, is in full progress. Membrane-bound signal transducers for growth factors are amongst the substances of interest. Gangliosides are lipid-sugar compounds localized on the cell membrane that are thought to modify pertinent signals and, therefore, may influence a variety of functions in normal and pathologic conditions including those that act upon tumor growth. Intracranial supratentorial astrocytic gliomas of the adult represent a tumor group, that may be divided into three grades of malignancy, the most anaplastic member being the glioblastoma. A stepwise anaplasia is assumed and accompanied by genetic events that are partly specific for these grades. In earlier investigations, it had been shown that there is a tendency towards formation of more simple members of the ganglioside family with ongoing malignancy of those tumors. Yet, the results were only partly congruent and the correlation to tumor grades rather loose. We, therefore, investigated the occurrence of triaose gangliosides within these tumors in situ by immunohistochemistry. In this paper, we corroborate our earlier observation that triaose gangliosides preferentially occur within the cytoplasm of large protoplasmic and gemistocytic astrocytes. The potency of the expression of GD2 is calculated and plotted against the expression of two markers of intermediate glial filaments, namely GFAP (glial fibrillary acid protein) and vimentine. A high interdependence of the three compounds could be demonstrated by correlation analysis. Thus, the conclusion must be drawn that the correlation of ganglioside patterns to the proliferation of astrocytic tumors is as poor as that of GFAP or vimentin expression, respectively.

TNFalpha-308A associated with shorter intervals of Plasmodium falciparum reinfections.

A mutation at position -308 of the tumor necrosis factor alpha (TNF-308A) gene promoter has previously been associated with particular manifestations of infectious and non-infectious diseases. In a longitudinal study on malariological parameters in Gabon, TNF promoter variants of 98 children initially presenting with severe Plasmodium falciparum malaria, followed by a total of 504 reinfection events within 52 months, and 100 children initially presenting with mild malaria followed by a total of 342 reinfections were analyzed. Symptomatic P. falciparum reinfections occurred more quickly (median 11 weeks) in carriers of the TNF-308A allele, with severe malaria at enrollment, compared to carriers of other TNF promoter variants (median 16 weeks). The results show that this particular TNF promoter allele increases the risk of reinfection with the malaria parasite P. falciparum.

Amodiaquine-artesunate versus amodiaquine for uncomplicated Plasmodium falciparum malaria in African children: a randomised, multicentre trial.

BACKGROUND: Increasing drug resistance limits the choice of efficacious chemotherapy against Plasmodium falciparum malaria in Africa. Amodiaquine still retains efficacy against P falciparum in many African countries. We assessed the safety, treatment efficacy, and effect on gametocyte carriage of adding artesunate to amodiaquine in three randomised trials in Kenya, Sénégal, and Gabon. METHODS: We enrolled 941 children (400 in Kenya, 321 in Sénégal, and 220 in Gabon) who were 10 years or older and who had uncomplicated P falciparum malaria. Patients were randomly assigned amodiaquine (10 mg/kg per day for 3 days) plus artesunate (4 mg/kg per day for 3 days) or amodiaquine (as above) and placebo (for 3 days). The primary endpoints were parasitological cure rates at days 14 and 28. Analysis was by intention to treat and by an evaluability method. FINDINGS: Both regimens were well tolerated. Six patients in the amodiaquine-artesunate group and five in the amodiaquine group developed early, drug-induced vomiting, necessitating alternative treatment. By intention-to-treat analysis, the day-14 cure rates for amodiaquine-artesunate versus amodiaquine were: 175/192 (91%) versus 140/188 (74%) in Kenya (D=16.7% [95% CI 9.3-24.1], p<0.0001), 148/160 (93%) versus 147/157 (94%) in Sénégal (-1.1% [-6.7 to 4.5], p=0.7), and 92/94 (98%) versus 86/96 (90%) in Gabon (8.3% [1.5-15.1], p=0.02). The corresponding rates for day 28 were: 123/180 (68%) versus 75/183 (41%) in Kenya (27.3% [17.5-37.2], p<0.0001), 130/159 (82%) versus 123/156 (79%) in Sénégal (2.9% [-5.9 to 11.7], p=0.5), and 80/94 (85%) versus 70/98 (71%) in Gabon (13.7% [2.2-25.2], p=0.02). Similar rates were obtained by evaluability analysis. INTERPRETATION: The combination of artesunate and amodiaquine improved treatment efficacy in Gabon and Kenya, and was equivalent in Sénégal. Amodiaquine-artesunate is a potential combination for use in Africa. Further investigations to assess the potential effect on the evolution of drug resistance, disease transmission, and safety of amodiaquine-artesunate are warranted.

Artesunate and praziquantel for the treatment of Schistosoma haematobium infections: a double-blind, randomized, placebo-controlled study.

Recently, artemisinin derivatives have been shown to be efficacious in chemoprophylaxis of and chemotherapy for Schistosoma japonicum and S. mansoni infections. Therefore, a double-blind, randomized, placebo-controlled study was carried out to investigate the efficacy and tolerability of artesunate plus placebo and the combination of artesunate and praziquantel in the treatment of S. haematobium infections in Gabon. The 300 infected schoolchildren included in the study were randomized to receive artesunate plus placebo (n=90), praziquantel plus placebo (n=90), artesunate and praziquantel (n=90), or only placebo (n=30). End points were efficacy, assessed as cure on day 56, and tolerability. All treatment regimens were well tolerated. The praziquantel plus placebo-treated group attained a cure rate of 73%, artesunate plus placebo a rate of 27%, the combination of artesunate and praziquantel a rate of 81%, and placebo alone a rate of 20%. In summary, earlier findings of efficacy of artemisinin derivitives against S. mansoni and S. japonicum could not be confirmed in S. haematobium infections.

Plasmodium falciparum transmission intensity and infection rates in children in Gabon.

Several factors can determine the outcome of a malarial infection. Studies on susceptibility or resistance to malarial infection can be confounded by differences in transmission. In the present study, the relationship between vector abundance and Plasmodium falciparum infection rate of Gabonese children was studied. Indoor human bait catches were conducted in the houses of two groups of children, those who had been found earlier to be either frequently (> 3 infections per year) or rarely (< 0.5 infections per year) infected with P. falciparum. The human biting rate was 12 and 31 bites per person per night during the dry and the rainy season, with 3% and 16% Anopheles, respectively. Anopheles gambiae and A. moucheti were found to be the only vectors involved in the transmission of malaria in this area. No significant difference in the abundance and the rate of P. falciparum infection of the Anopheles mosquitoes was found among children rarely or frequently infected. Differences in transmission cannot account for differences in infection rates in our study group. Hereditary and immunological factors seem to be the primary determinants for the outcome of malarial infection.

Plasmodium falciparum: in vitro growth inhibition by febrile temperatures.

Febrile episodes are the hallmark of malarial infection. We determined the inhibitory effect of febrile temperatures on the in vitro growth of Plasmodium falciparum. Parasites were cultured at various temperatures between 37 degrees C and 40 degrees C for 4 days. A logistic decrease in parasitaemia as a function of temperature was observed for continuous cultures. Incubation of synchronized cultures for different lengths of time during the parasite cycle showed a strong increase of growth inhibition with the maturing of parasites. Febrile temperatures inhibit parasite growth and long, high fevers during malaria may be beneficial for parasite clearance.

Effect of antipyretic drugs in children with malaria.

A comparison of different antipyretics in children with malaria showed a small effect of naproxen, but not of metamizol, on the reduction of fever peaks. Antipyretic treatment had no effect on fever clearance and therefore should be used cautiously in the treatment of malaria.

HLA-DQB1*0501-restricted Th1 type immune responses to Plasmodium falciparum liver stage antigen 1 protect against malaria anemia and reinfections.

Protective immunity against Plasmodium falciparum requires constant exposure to the pathogen. T cell-mediated immune responses are induced by T cell epitopes of pre-erythrocytic stage antigens of P. falciparum and involve HLA-restricted CD4 and CD8 cells. Cytotoxic T cell responses to a conserved epitope of P. falciparum liver stage antigen (LSA) type 1 are restricted by the HLA class I allele Bw53. The role of HLA class II alleles in mediating cellular responses against P. falciparum LSA-1 has not yet been demonstrated. In a longitudinal study performed for >4 years, associations were found between the HLA class II allele DQB1*0501 and protection from malaria anemia and malarial reinfections in Gabonese children. Children carrying DQB1*0501 had a higher frequency of interferon-gamma responses to LSA-1 T cell epitopes, compared with noncarriers.

Atopy and malaria.

The last decades have seen a dramatic rise in the prevalence of allergic diseases throughout the industrialised world. The "hygiene hypothesis" postulates that this is due to a reduced exposure to infections during childhood. A cohort study in children from Gabon gave us the unique opportunity to examine the relationship between exposure to P. falciparum and atopy. 91 children, who had been closely followed for an average of 5 years and of whom the exact incidence of malaria attacks was known, underwent a skin-prick test with mite antigen. 16 children (18%) had a positive reaction. Gender or age had no effect on the outcome of the test. However, those tested positive had had less infections and a lower incidence of malaria than children tested negative (p = 0.017). Survival analysis shows that children with a high exposure to P. falciparum were at lower risk of an atopic skin reaction (p = 0.001). We postulate that the low exposure to the malaria parasite contributes to the development of an imbalanced immune system with a subsequent higher reactivity to the allergen tested. Immuno-suppression is commonly seen during a malaria attack and this correlates positively with the level of anti-inflammatory cytokines such as interleukin-10. High exposure to parasite antigens might counterbalance pro-inflammatory immune reactions and thus protect against allergic diseases. A better understanding of the relationship between parasitic infection and allergy will help us to develop strategies to prevent allergic disease without being exposed to infectious diseases.

Change in Plasmodium falciparum genotype during successive malaria episodes in Gabonese children.

Extensive polymorphism in the malaria parasite Plasmodium falciparum is one of the major obstacles to controlling the disease. With the aim of analysing the dynamics of P. falciparum inoculations, we investigated the parasite genotypes of successive malaria episodes. Polymerase chain reaction was performed on blood samples collected longitudinally from 31 children in Lambaréné, Gabon. The polymorphic regions of the merozoite surface antigens 1 and 2 were used as genetic markers. The data show that children in this area are exposed to many different P. falciparum strains. In a few cases, the same parasite genotypic pattern was observed in samples from two consecutive clinical attacks indicating probable recrudescences after therapy. In six cases the first successive infections with a particular merozoite surface antigen (MSA)-2 strain (3D7) were followed by infections with the other MSA-2 genotype (FC27). In all other cases the genetic characteristics of the parasite were different from one infection to the next, indicating that reinfection was caused by a new parasite strain.

Decreased atopy in children infected with Schistosoma haematobium: a role for parasite-induced interleukin-10.

BACKGROUND: Most of the effort directed at understanding the role infections have in preventing allergy has focused on bacteria and viruses and their ability to divert the immune system towards T-helper-1 responses and away from proallergic T-helper-2 responses. However, helminth infections, highly prevalent in large parts of the developing world, where allergy is uncommon, stimulate strong T-helper-2 responses. We investigated the influence of chronic helminth infections on the prevalence of atopy and aimed to understand the relation at a detailed immunological level. METHODS: 520 Gabonese schoolchildren were tested for skin reaction to house-dust mite and other allergens, for Schistosoma haematobium eggs in urine, and for microfilariae in blood samples. Total and mite-specific IgE antibodies were measured. A subsample selected on the basis of their skin test to house-dust mite received detailed immunological investigations. FINDINGS: Children with urinary schistosomiasis had a lower prevalence of a positive skin reaction to house-dust mite than those free of this infection (odds ratio 0.32 [95% CI 0.16-0.63]). The degree of sensitisation to house-dust mite could not explain this difference in skin-prick positivity. Schistosome-antigen-specific concentrations of interleukin-10 were significantly higher in infected children, and higher specific concentrations of this anti-inflammatory cytokine were negatively associated with the outcome of skin-test reactivity to mite (0.53 [0.30-0.96]). No association between polyclonal IgE antibodies and skin-test results was found. INTERPRETATION: The anti-inflammatory cytokine, interleukin-10, induced in chronic schistosomiasis, appears central to suppressing atopy in African children.