The value of and challenges for cholera vaccines in Africa.

The 21st century saw a shift in the cholera burden from Asia to Africa. The risk factors for cholera outbreaks in Africa are incompletely understood, and the traditional emphasis on providing safe drinking water and improving sanitation and hygiene has proven remarkably insufficient to contain outbreaks. Current killed whole-cell oral cholera vaccines (OCVs) are safe and guarantee a high level of protection for several years. OCVs have been licensed for >20 years, but their potential for preventing and control cholera outbreaks in Africa has not been realized. Although each item in the long list of technical reasons why cholera vaccination campaigns have been deferred is plausible, we believe that the biggest barrier is that populations affected by cholera outbreaks are underprivileged and lack a strong political voice. The evaluation and use of OCVs as a tool for cholera control will require a new, more compassionate, less risk-averse generation of decision makers.

Green Synthesis of Silver Nanoparticle from Anadenanthera colubrina Extract and Its Antimicrobial Action against ESKAPEE Group Bacteria.

Given the urgent need for novel methods to control the spread of multidrug-resistant microorganisms, this study presents a green synthesis approach to produce silver nanoparticles (AgNPs) using the bark extract from Anadenanthera colubrina (Vell.) Brenan var. colubrina. The methodology included obtaining the extract and characterizing the AgNPs, which revealed antimicrobial activity against MDR bacteria. A. colubrina species is valued in indigenous and traditional medicine for its medicinal properties. Herein, it was employed to synthesize AgNPs with effective antibacterial activity (MIC = 19.53-78.12 µM) against clinical isolates from the ESKAPEE group, known for causing high hospitalization costs and mortality rates. Despite its complexity, AgNP synthesis is an affordable method with minimal environmental impacts and risks. Plant-synthesized AgNPs possess unique characteristics that affect their biological activity and cytotoxicity. In this work, A. colubrina bark extract resulted in the synthesis of nanoparticles measuring 75.62 nm in diameter, with a polydispersity index of 0.17 and an average zeta potential of -29 mV, as well as low toxicity for human erythrocytes, with a CC50 value in the range of 961 µM. This synthesis underscores its innovative potential owing to its low toxicity, suggesting applicability across several areas and paving the way for future research.

Synthesis of Silver Nanoparticles Using Extracts from Different Parts of the Paullinia cupana Kunth Plant: Characterization and In Vitro Antimicrobial Activity.

The green synthesis of silver nanoparticles (AgNPs) can be developed using safe and environmentally friendly routes, can replace potentially toxic chemical methods, and can increase the scale of production. This study aimed to synthesize AgNPs from aqueous extracts of guarana (Paullinia cupana) leaves and flowers, collected in different seasons of the year, as a source of active biomolecules capable of reducing silver ions (Ag+) and promoting the stabilization of colloidal silver (Ag0). The plant aqueous extracts were characterized regarding their metabolic composition by liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS), phenolic compound content, and antioxidant potential against free radicals. The synthesized AgNPs were characterized by UV/Vis spectrophotometry, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and scanning electron microscopy coupled to energy-dispersive X-ray spectrometry (EDX). The results demonstrated that the chemical characterization indicated the presence of secondary metabolites of many classes of compounds in the studied aqueous extracts studied, but alkaloids and flavonoids were predominant, which are widely recognized for their antioxidant capabilities. It was possible to notice subtle changes in the properties of the nanostructures depending on parameters such as seasonality and the part of the plant used, with the AgNPs showing surface plasmon resonance bands between 410 and 420 nm using the leaf extract and between 440 and 460 nm when prepared using the flower extract. Overall, the average hydrodynamic diameters of the AgNPs were similar among the samples (61.98 to 101.6 nm). Polydispersity index remained in the range of 0.2 to 0.4, indicating that colloidal stability did not change with storage time. Zeta potential was above -30 mV after one month of analysis, which is adequate for biological applications. TEM images showed AgNPs with diameters between 40.72 to 48.85 nm and particles of different morphologies. EDX indicated silver content by weight between 24.06 and 28.81%. The synthesized AgNPs exhibited antimicrobial efficacy against various pathogenic microorganisms of clinical and environmental interest, with MIC values between 2.12 and 21.25 µg/mL, which is close to those described for MBC values. Therefore, our results revealed the potential use of a native species of plant from Brazilian biodiversity combined with nanotechnology to produce antimicrobial agents.

Diagnosis, clinical presentation, and in-hospital mortality of severe malaria in HIV-coinfected children and adults in Mozambique.

BACKGROUND: Severe falciparum malaria with human immunodeficiency virus (HIV) coinfection is common in settings with a high prevalence of both diseases, but there is little information on whether HIV affects the clinical presentation and outcome of severe malaria. METHODS: HIV status was assessed prospectively in hospitalized parasitemic adults and children with severe malaria in Beira, Mozambique, as part of a clinical trial comparing parenteral artesunate versus quinine (ISRCTN50258054). Clinical signs, comorbidity, complications, and disease outcome were compared according to HIV status. RESULTS: HIV-1 seroprevalence was 11% (74/655) in children under 15 years and 72% (49/68) in adults with severe malaria. Children with HIV coinfection presented with more severe acidosis, anemia, and respiratory distress, and higher peripheral blood parasitemia and plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP2). During hospitalization, deterioration in coma score, convulsions, respiratory distress, and pneumonia were more common in HIV-coinfected children, and mortality was 26% (19/74) versus 9% (53/581) in uninfected children (P < .001). In an age- and antimalarial treatment-adjusted logistic regression model, significant, independent predictors for death were renal impairment, acidosis, parasitemia, and plasma PfHRP2 concentration. CONCLUSIONS: Severe malaria in HIV-coinfected patients presents with higher parasite burden, more complications, and comorbidity, and carries a higher case fatality rate. Early identification of HIV coinfection is important for the clinical management of severe malaria.

Effectiveness of mass oral cholera vaccination in Beira, Mozambique.

BACKGROUND: New-generation, orally administered cholera vaccines offer the promise of improved control of cholera in sub-Saharan Africa. However, the high prevalence of human immunodeficiency virus (HIV) infection in many cholera-affected African populations has raised doubts about the level of protection possible with vaccination. We evaluated a mass immunization program with recombinant cholera-toxin B subunit, killed whole-cell (rBS-WC) oral cholera vaccine in Beira, Mozambique, a city where the seroprevalence of HIV is 20 to 30 percent. METHODS: From December 2003 to January 2004, we undertook mass immunization of nonpregnant persons at least two years of age, using a two-dose regimen of rBS-WC vaccine in Esturro, Beira (population 21,818). We then assessed vaccine protection in a case-control study during an outbreak of El Tor Ogawa cholera in Beira between January and May 2004. To estimate the level of vaccine protection, antecedent rates of vaccination were compared between persons with culture-confirmed cholera severe enough to have prompted them to seek treatment and age- and sex-matched neighborhood controls without treated diarrhea. RESULTS: We assessed the effectiveness of the vaccine in 43 persons with cholera and 172 controls. Receipt of one or more doses of rBS-WC vaccine was associated with 78 percent protection (95 percent confidence interval, 39 to 92 percent; P=0.004). The vaccine was equally effective in children younger than five years of age and in older persons. A concurrently conducted case-control study designed to detect bias compared persons with treated, noncholeraic diarrhea and controls without diarrhea in the same population and found no protection associated with receipt of the rBS-WC vaccine. CONCLUSIONS: The rBS-WC vaccine was highly effective against clinically significant cholera in an urban sub-Saharan African population with a high prevalence of HIV infection.

Is HIV infection associated with an increased risk for cholera? Findings from a case-control study in Mozambique.

OBJECTIVE: As residents of sub-Saharan Africa are at high risk for HIV and cholera, it is biologically plausible that immune suppression caused by HIV infection predisposes to cholera. Our aim was to assess the potential association between both diseases. METHODS: We conducted a case-control study in Beira, Mozambique, a high-risk area for HIV and cholera. Between 1 January 2005 and 30 June 2006, experienced counsellors invited 132 suspected cholera cases and 528 age- and sex-matched controls to an HIV counselling and testing centre. RESULTS: Forty (30%) of the invited cases and 127 (24%) of the invited controls came for HIV testing. No significant differences in demographic and socio-economic baseline characteristics were detected between participants and non-participants. Twenty five of 167 (15%) individuals who underwent testing were found HIV-positive. The probability of a positive HIV-test was highest in participants between 40 and 49 years; 6 of 14 (43%) tested HIV-positive. Nine of 40 (23%) cholera cases were found to be HIV-infected compared with 16 of 127 (13%) controls (adjusted odds ratio 2.6; 95% CI 0.9-7.5; P = 0.08). DISCUSSION: The findings suggest that in a cholera-endemic area, HIV infection is associated with an increased risk for cholera. More research in HIV endemic settings is needed to confirm the findings and to explore the effect of HIV-related immunosuppression on the transmission of cholera.

Characteristics of a pandemic clone of O3 : K6 and O4 : K68 Vibrio parahaemolyticus isolated in Beira, Mozambique.

The genetic characteristics of Vibrio parahaemolyticus strains isolated in 2004 and 2005 in Mozambique were assessed in this study to determine whether the pandemic clone of V. parahaemolyticus O3 : K6 and O4 : K68 serotypes has spread to Mozambique. Fifty-eight V. parahaemolyticus strains isolated from hospitalized diarrhoea patients in Beira, Mozambique, were serotyped for O : K antigens and genotyped for toxR, tdh and trh genes. A group-specific PCR, a PCR that detects the presence of ORF8 of the filamentous phage f237, arbitrarily primed PCR, PFGE and multilocus sequence typing were performed to determine the pandemic status of the strains and their ancestry. All strains of serovars O3 : K6 (n=38) and O4 : K68 (n=4) were identified as a pandemic clonal group by these analyses. These strains are closely related to the pandemic reference strains of O3 : K6 and O4 : K68, which emerged in Asia in 1996 and were later found globally. The pandemic serotypes O3 : K6 and O4 : K68 including reference strains grouped into a single cluster indicating emergence from a common ancestor. The O3 : K58 (n=8), O4 : K13 (n=6), O3 : KUT (n=1) and O8 : K41 (n=1) strains showed unique characteristics different from the pandemic clone.

Multilocus sequence typing (MLST) analysis of Vibrio cholerae O1 El Tor isolates from Mozambique that harbour the classical CTX prophage.

Vibrio cholerae O1 isolates belonging to the Ogawa serotype, El Tor biotype, harbouring the classical CTX prophage were first isolated in Mozambique in 2004. Multilocus sequence typing (MLST) analysis using nine genetic loci showed that the Mozambique isolates have the same sequence type (ST) as O1 El Tor N16961, a representative of the current seventh cholera pandemic. Analysis of the CTX prophage in the Mozambique isolates indicated that there is one type of rstR in these isolates: the classical CTX prophage. It was also found that the ctxB-rstR-rstA-rstB-phs-cep fragment was PCR-amplified from these isolates, which indicates the presence of a tandem repeat of the classical CTX prophage in the genome of the Mozambique isolates. The possible origin of these isolates and the presence of the tandem repeat of the classical prophage in them implicate the presence of the classical CTX phage.

Field evaluation of a rapid immunochromatographic dipstick test for the diagnosis of cholera in a high-risk population.

BACKGROUND: Early detection of cholera outbreaks is crucial for the implementation of the most appropriate control strategies. METHODS: The performance of an immunochromatographic dipstick test (Institute Pasteur, Paris, France) specific for Vibrio cholerae O1 was evaluated in a prospective study in Beira, Mozambique, during the 2004 cholera season (January-May). Fecal specimens were collected from 391 patients with acute watery nonbloody diarrhea and tested by dipstick and conventional culture. RESULTS: The overall sensitivity and specificity of the rapid test compared to culture were 95% (95% confidence interval [CI]: 91%-99%) and 89% (95% CI: 86%-93%), respectively. After stratification by type of sample (rectal swab/bulk stool) and severity of diarrhea, the sensitivity ranged between 85% and 98% and specificity between 77% and 97%. CONCLUSION: This one-step dipstick test performed well in the diagnosis of V. cholerae O1 in a setting with seasonal outbreaks where rapid tests are most urgently needed.

The high burden of cholera in children: comparison of incidence from endemic areas in Asia and Africa.

BACKGROUND: Cholera remains an important public health problem. Yet there are few reliable population-based estimates of laboratory-confirmed cholera incidence in endemic areas around the world. METHODS: We established treatment facility-based cholera surveillance in three sites in Jakarta (Indonesia), Kolkata (India), and Beira (Mozambique). The annual incidence of cholera was estimated using the population census as the denominator and the age-specific number of cholera cases among the study cohort as the numerator. FINDINGS: The lowest overall rate was found in Jakarta, where the estimated incidence was 0.5/1000 population/year. The incidence was three times higher in Kolkata (1.6/1000/year) and eight times higher in Beira (4.0/1000/year). In all study sites, the greatest burden was in children under 5 years of age. CONCLUSION: There are considerable differences in cholera incidence across these endemic areas but in all sites, children are the most affected. The study site in Africa had the highest cholera incidence consistent with a growing impression of the large cholera burden in Africa. Burden estimates are useful when considering where and among whom interventions such as vaccination would be most needed.

Private demand for cholera vaccines in Beira, Mozambique.

In the summer of 2005, we interviewed 996 randomly selected respondents in Beira, Mozambique concerning their willingness and ability to pay for cholera vaccine for themselves and for other household members. Respondents were told that two doses of the vaccine would be required 2 weeks apart, and that the cholera vaccine would offer excellent protection against infection for the first year following vaccination, and some protection during the second and third year after a person is vaccinated. This research was carried out in order to learn more about private demand for vaccines in a cholera-endemic area. We asked two types of valuation questions: (1) a discrete-price offer for a vaccine that could be purchased for household members and (2) a payment card designed to assess uncertainty in the respondent's demand for a vaccine for self-protection. We estimate average household willingness to pay (WTP) for cholera vaccines in Beira to be 2005 US$ 8.45. This estimate of household WTP represents the perceived private economic benefits to a household--six persons on average--of giving all members free cholera vaccines.

Genetic diversity of El Tor strains of Vibrio cholerae O1 with hybrid traits isolated from Bangladesh and Mozambique.

Vibrio cholerae O1 strains that are hybrids between the classical and El Tor biotypes were isolated during two consecutive years (2004-2005) from diarrheal patients in Mozambique. Similar variants isolated in Bangladesh and recently isolated El Tor strains were analyzed for genetic diversity. Pulsed-field gel electrophoresis (PFGE) analysis using the restriction enzyme NotI, resulted in 18-21 bands showed five closely related PFGE patterns that were distributed similarly in both years (2004-2005) among the 80 strains tested in Mozambique. Overall based on the PFGE patterns the hybrids indicated an El Tor lineage. The restriction patterns of whole-chromosomal DNA grouped the hybrid strains from Mozambique into a separate cluster from Bangladeshi clinical and environmental hybrid strains. A high molecular weight band of 398kb that contain rstR allele of the classical type was detected from all hybrid strains, which was absent in all conventional classical and El Tor strains. This band could be designated as a marker for the hybrid strains. This study suggests that hybrid strains from Mozambique are closely related to each other, different from Bangladeshi hybrid strains that are diverse in nature and all hybrid strains differed markedly from conventional classical and El Tor strains.

Are rapid population estimates accurate? A field trial of two different assessment methods.

Emergencies resulting in large-scale displacement often lead to populations resettling in areas where basic health services and sanitation are unavailable. To plan relief-related activities quickly, rapid population size estimates are needed. The currently recommended Quadrat method estimates total population by extrapolating the average population size living in square blocks of known area to the total site surface. An alternative approach, the T-Square, provides a population estimate based on analysis of the spatial distribution of housing units taken throughout a site. We field tested both methods and validated the results against a census in Esturro Bairro, Beira, Mozambique. Compared to the census (population: 9,479), the T-Square yielded a better population estimate (9,523) than the Quadrat method (7,681; 95% confidence interval: 6,160-9,201), but was more difficult for field survey teams to implement. Although applicable only to similar sites, several general conclusions can be drawn for emergency planning.

Feasibility of a mass vaccination campaign using a two-dose oral cholera vaccine in an urban cholera-endemic setting in Mozambique.

We conducted a study to assess the feasibility and the potential vaccine coverage of a mass vaccination campaign using a two-dose oral cholera vaccine in an urban endemic neighbourhood of Beira, Mozambique. The campaign was conducted from December 2003 to January 2004. Overall 98,152 doses were administered, and vaccine coverage of the target population was 58.6% and 53.6% for the first and second rounds, respectively. The direct cost of the campaign, which excludes the price of the vaccine, amounted to slightly over 90,000 dollars, resulting in the cost per fully vaccinated person of 2.09 dollars, which is relatively high. However, in endemic settings where outbreaks are likely to occur, integrating cholera vaccination into the routine activities of the public health system could reduce such costs.

Pandemic serovars (O3:K6 and O4:K68) of Vibrio parahaemolyticus associated with diarrhea in Mozambique: spread of the pandemic into the African continent.

Forty-two episodes of Vibrio parahaemolyticus infections were detected in Beira, Mozambique, from January to May 2004. The majority of the isolates (81%) belonged to the pandemic serovars (O3:K6 and O4:K68) of V. parahaemolyticus. The pandemic serovars were positive by group-specific PCR (GS-PCR) and a PCR specific for open reading frame ORF8 (ORF8-PCR), which are molecular markers of the pandemic clone, and were positive for tdh but negative for trh. The remaining 19% of the strains also possessed the tdh gene but were GS-PCR and ORF8-PCR negative and did not belong to the pandemic serovars. Patients with V. parahaemolyticus infection were older (mean age, 27 years) than patients infected by other diarrheal agents (mean age, 21 years). Ten percent of diarrhea patients from whom no V. parahaemolyticus was cultured were severely dehydrated, but none of the V. parahaemolyticus cases were severely dehydrated. This is the first report of the isolation of pandemic strains of V. parahaemolyticus in sub-Saharan Africa and clearly indicates that the pandemic of V. parahaemolyticus has spread into the African continent.