Absence of hybridization with the wild-type and mutant rpoB probes in the Genotype MTBDRplus assay detects 'disputed' rifampicin mutations.

OBJECTIVE: Mycobacterium tuberculosis isolates that fail to hybridize to at least one rpoB wild-type or any mutation probe on the Genotype MTBDRplus strip are assumed to be rifampicin-resistant. However, the precise mutation(s) are unknown. We sought to identify the mutations in isolates with such hybridization patterns and determine if the mutations are associated with resistance to rifampicin. METHODS: In this study, 275 M. tuberculosis isolates were screened with the Genotype MTBDRplus assay to identify isolates with the hybridization pattern. These isolates were sequenced and their minimum inhibitory concentrations (MIC) determined using the Bactec MGIT 960 system. RESULTS: Among the 275 isolates tested, 15 (6%) isolates with the hybridization pattern were identified. Sequencing showed that failure to hybridize to rpoB wild-type probes resulted from the presence of 'disputed' rifampicin mutations, which are mutations not always associated with a rifampicin-resistant phenotype. All, except 3/15, isolates had a rifampicin-resistant phenotype (MIC > 1 µg/mL). One of the three isolates with a rifampicin-susceptible phenotype had the same mutation at position 526 (His526Leu) as another isolate that had a rifampicin-resistant phenotype. CONCLUSION: The recommendation of the Genotype MTBDRplus assay to assume rifampicin resistance based solely on failure to hybridize to rpoB wild-type probe allows the identification of important RIF-resistant isolates. About 20% (3/15) of such isolates could be missed by relying only on the standard MGIT 960 DST assay for drug susceptibility testing.

[Efficacy and safety of antimalarial combinations for treatment of uncomplicated malaria in children in Bangui, Central African Republic]. / Efficacité et tolérance des associations d'antipaludiques dans le traitement du paludisme simple chez les enfants à Bangui, République centrafricaine.

OBJECTIVE: The aim of this study was to evaluate the efficacy and safety of three anti-malarial combinations--artemether-lumefantrine (A-L), amodiaquine-sulfadoxine-pyrimethamine (AQ-SP), and artesunate-amodiaquine (AQ-AS)--in the treatment of uncomplicated malaria in children younger than 5 years in Bangui, Central African Republic. METHODOLOGY: This study included 186 children aged 6-59 months with uncomplicated falciparum malaria who were treated at the Bédé Combattant Hospital from July through October 2010: 63 randomized to receive A-L, 63 AQ-SP, and 60 AQ-AS. Clinical outcome was classified according to WHO criteria as early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF), or adequate clinical and parasitological response (ACPR). The occurrence of mutations in the pfcrt, pfmdr-1, dhfr and dhps genes was studied by PCR-RFLP. RESULTS: After PCR correction, ACPR at D28 was 100% for A-L, 96.55% for AQ-SP, and 100% for AQ-AS, with no significant difference between the three combinations (p = 0.36). The 2 cases of treatment failure for AQ-SP were associated with mutations at the following resistance markers: Pfcrt 76T, PfmdrI 86Y, Dhfr 108N, and Dhps A437. There was no significant difference in the reduction of anemia, fever (p = 0.87), or parasitemia (p = 0.63) between the three combinations. CONCLUSION: This study demonstrates that artemisinin-based combinations are still effective and tolerated in the treatment of uncomplicated malaria in children younger than 5 years in Bangui. Treatment failures were due to new infections and mutations in resistance markers.

Medicine sellers' perspectives on their role in providing health care in North-West Cameroon: a qualitative study.

BACKGROUND: Increasing recognition of the importance of medicine sellers in low-resource settings has emerged alongside assumptions that their motives and capacities primarily relate to profit maximization. This article suggests a need to reframe thinking about the role of medicine sellers in developing country health systems. METHODS: We used in-depth interviews to explore perceptions of medicine seller roles among a restricted random sample of 20 medicine sellers in North-West Cameroon. Interviews and analysis explored self-perception of their work/role, community perceptions, skills and knowledge, regulation, future plans, links with the formal health system and diversity among medicine sellers. RESULTS: Medicine sellers in our study were a varied, yet distinct group. They saw themselves as closely integrated in the social and medical landscapes of clients. Although some client interactions were described as simple sales, many respondents presented themselves as gatekeepers of medicines and knowledge, reflecting a conceptualization of the distinctness of medicines over other commodities. Acknowledgement of limits in knowledge and resources led to recognition of the need for formal healthcare providers and justified a restricted scope of practice and the need for referral. Motivation was derived from a desire for both financial and social capital combined with a proximity to medicines and repeated exposure to ill health. Legitimacy was perceived to be derived from: a historical mandate; informal and formal training and effective 'community regulation'. CONCLUSIONS: The distinct role that medicine sellers describe themselves as occupying in this study area can be characterized as provision of 'first aid', urgent, reactive and sometimes providing intermediate care prior to referral. Medicine sellers suggest that they do not aspire to be doctors and emphasize the complementary, rather than competitive, nature of their relationship with formal providers. We discuss the challenges and opportunities of characterizing medicine sellers as a distinctive group of 'first aiders' in this setting.

[Molecular markers for malaria drug resistance: necessary but not sufficient criteria to decide change in treatment policy]. / Marqueurs moléculaires de résistance aux médicaments antipaludiques: nécessaires mais pas suffisants pour un changement de politique de santé.

Molecular markers or gene mutations that are associated with resistance have been the recent focus for an attempt to promptly determine the establishment of resistance to known and currently used antimalaria drugs. For control managers, the effective management of malaria would involve strategies of interruption of the malaria transmission and/or improved therapeutic management of malaria. To place molecular markers within the context of control programs requires that one recognises the two data pools necessary for effective evidence-based policy change. These include data on socio-economic determinants on the one hand and biomedical data on the other. The markers for clinical efficacy of drugs have principally been genes either associated with transport or metabolism of the drug. In malaria those that have been the most characterised are the Pfcrt, Pfmdrl for the quinolines and the dhfr and dhps genes for the anti-folates. The PfATPase has been suggested to be involved in the recently developed artermisinine based combination therapies (ACT). To consider changes in drug policy, a control manager needs to address: efficacy, transmissibility, disease dynamics, safety, epidemics, tolerability and compliance. Except for safety and tolerability/compliance, molecular markers do provide useful information. However these markers still have to be validated alongside in vitro studies and in many different ecological settings and shown to be stable over time or associated with changing drug efficacy situations. Besides the evidence provided with these tools, the government will be required to ensure a mass education of the population and care providers, and fight against illicit street vendors. The governments will therefore still wary on the resources necessary to occasion an effective switch in drug policy especially at the district level and in the rural areas where meaningful, cost-effective programs are most needed.

Molecular characterisation of Pseudomonas aeruginosa recovered in the Buea health district of Cameroon: implications for nosocomial spread.

BACKGROUND: Pseudomonas aeruginosa is a ubiquitous gram-negative pathogen with a propensity to cause opportunistic infections in humans. Different strains of the organism could colonise patients heralding a wide spectrum of P. aeruginosa infections in the environment. OBJECTIVE: To analyse isolates of P. aeruginosa from clinical and environmental samples using the Polymerase Chain Reaction (PCR) to establish strain relatedness. METHODS: Fifty-two strains of the organism were isolated from wound swabs, urine, sputum of patients and environmental samples from the hospital environment using standard microbiological techniques and ethical consideration. Genomic DNA of the isolates was amplified with primers AF1 (5'-AGA GTT TGA TCC TGG CTCA-3') and 1541R (5'-AAG GAG GTG ATC CAG CC-3'). RESULTS: At least two bands were observed in all isolates typed and band sizes ranged from 0.07 - 1.5kb. The strains were genetically diverse, displaying profiles of 2 - 6 bands between 0.07 - 1.5kb. CONCLUSION: The study demonstrates that strain diversity could be discerned between strains of P. aeruginosa, circulating in the environment of Buea, a finding which has important epidemiological and clinical significance bearing in mind that this pathogen is highly incriminated in nosocomial infections with attendant social implications. This therefore calls for more attention in the diagnosis and management of P. aeruginosa infections in the environment of Buea, Cameroon.

Seasonal variation and prevalence of tuberculosis among health seekers in the South Western Cameroon.

OBJECTIVES: To determine the prevalence of tuberculosis (TB) in Fako health District, to assess the effects of seasonal variation on the incidence of TB in the study area and to use sentinel analysis to predict areas of greatest infection. DESIGN: A prospective cross sectional study based on laboratory investigations. SETTING: Fako health District, South Western Cameroon. RESULTS: The prevalence of TB was 23.3%. Tuberculosis was significantly more prevalent in males (12.6%) as compared with females (10.7%) (P = 0.034). TB prevalence was significantly different between age groups, with the highest number of cases recorded in the age group 21-30 (P = 0.002). When the health areas were compared, TB prevalence varied significantly (P = 0.001), with Limbe Town recording the highest number of TB cases. We recorded more TB cases in the wet season compared with the dry season and the difference was statistically significant (P = 0.000). There was a significant drop in the prevalence of TB over the study period (P = 0.000). CONCLUSION: This study is the first to report on the effects of season on the prevalence of TB in Cameroon. These findings will therefore provide additional useful base line data for setting up TB control strategies in Cameroon.

Antibiogram and plasmid profiles of Neisseria gonorrhoeae isolates from Cameroon: useful tools for epidemiological survey.

A prospective laboratory-based investigative study was carried out on clinical isolates of N. gonorrhoea to determine their antibiotic susceptibility patterns and plasmid profile using standard microbiological and molecular techniques. All the 32 isolates studied showed total resistance to penicillin, spectinomycin and amoxyclinE. On the other hand, susceptibilities of 100%, 98.6% and 98.6% were noted for ciprofloxacin, ofloxacin and norfloxacin respectively. Thirty (93.8%) of the 32 isolates were found to harbour plasmids of molecular weights ranging from 9.2 to 25.2 Mdal. Three distinct groups of N. gonorrhoea isolates were identified based on the molecular weights of the plasmids, namely, group one (9.2 Mdal), group two (12.6 Mdal) and group three (25.2 Mdal). These results suggest that different strains of N. gonorrhoea may be circulating in Fako Division of Cameroon, a finding that is of clinical and epidemiological significance.

Antibiogram of Klebsiella pneumoniae isolates from Buea, Cameroon.

OBJECTIVE: To determine the antibiotic susceptibility of K. pneumoniae isolates from Buea, Cameroon. DESIGN: A prospective study of K. pneumoniae isolates from clinical samples of nosocomial origin. SETTING: A laboratory based investigative study at the Biotechnology Centres of the Universities of Buea and Yaounde 1, Cameroon, and three Buea based hospitals. K. pneumoniae isolates were obtained from sputum, wound swabs and urine and screened for their antibiogram using standard procedures. RESULTS: Results on the antibiogram showed seven distinct antibiotypes distinguished by different susceptibilities to aminoglycosides (Spectinomycin and Gentamicin), Chloramphenicol and Augmentin. All the isolates shared multi-resistance to Amoxicillin and Trimethoprim. However, the isolates showed marked susceptibilities to Norfloxacin (90.01%), Cefuroxime (95.45%) and Ciprofloxacin (86.36%). CONCLUSION: The study has revealed that K. pneumoniae isolates in the environment of Buea, Cameroon are multi-drug resistant. This finding is of clinical and epidemiological significance.

3' UTR elements enhance expression of Pgs28, an ookinete protein of Plasmodium gallinaceum.

In Plasmodium parasites the fusion of gametes to form a fertilized zygote and morphogenesis into the motile ookinete are critical developmental stages in the parasite's complex life cycle. In analogous developmental stages of metazoan organisms 3' gene flanking regions are critical in the regulation of gene expression. To determine whether these mechanisms are conserved in the protozoan parasite we studied the 3' gene flanking elements necessary for the expression of Pgs28, the major surface protein of mature zygotes and ookinetes of the chicken malaria Plasmodium gallinaceum. The DNA sequence of the pgs28 3' gene flanking region contains 7 eukaryotic polyadenylation consensus signals (AATAAA/ATTAAA). An unusual 82% T-rich region is located 55 nucleotides upstream of the fifth polyadenylation signal (ATTAAA). The pgs28 mRNA terminates approximately 20 nucleotides from the polyadenylation signal in a poly (A) tail. To determine whether the T-rich region and polyadenylation signals were necessary for Pgs28 protein expression, sexual stage parasites were transfected with plasmids containing deletions of these elements utilizing firefly luciferase (LUC) and beta-glucuronidase (GUS) as markers of transient gene transfection. The parasites were allowed to develop in vitro to the ookinete stage and assayed for enzymatic activity. Cells transfected with plasmids containing deletions of the T-rich region or fifth eukaryotic polyadenylation consensus signal expressed 89 and 92%, less enzymatic activity respectively than those transfected with the full length pgs28 3' gene flanking region. The U-rich element and fifth eukaryotic polyadenylation consensus sequence within the pgs28 3' UTR are therefore necessary for Pgs28 protein expression.

Isolation and functional characterization of two distinct sexual-stage-specific promoters of the human malaria parasite Plasmodium falciparum.

Transmission of malaria depends on the successful development of the sexual stages of the parasite within the midgut of the mosquito vector. The differentiation process leading to the production of the sexual stages is delineated by several developmental switches. Arresting the progression through this sexual differentiation pathway would effectively block the spread of the disease. The successful development of such transmission-blocking agents is hampered by the lack of a detailed understanding of the program of gene expression that governs sexual differentiation of the parasite. Here we describe the isolation and functional characterization of the Plasmodium falciparum pfs16 and pfs25 promoters, whose activation marks the developmental switches executed during the sexual differentiation process. We have studied the differential activation of the pfs16 and pfs25 promoters during intraerythrocytic development by transfection of P. falciparum and during gametogenesis and early sporogonic development by transfection of the related malarial parasite P. gallinaceum. Our data indicate that the promoter of the pfs16 gene is activated at the onset of gametocytogenesis, while the activity of the pfs25 promoter is induced following the transition to the mosquito vector. Both promoters have unusual DNA compositions and are extremely A/T rich. We have identified the regions in the pfs16 and pfs25 promoters that are essential for high transcriptional activity. Furthermore, we have identified a DNA-binding protein, termed PAF-1, which activates pfs25 transcription in the mosquito midgut. The data presented here shed the first light on the details of processes of gene regulation in the important human pathogen P. falciparum.

A monoclonal antibody-based immunodiagnostic assay for onchocerciasis.

Five murine monoclonal antibodies raised against Onchocerca volvulus cuticular extracts and termed MOVs (1-4 and 6) were selected based on reactivity with O. volvulus cryosections, and non-reactivity with cryosections of human skin and/or nodular tissue. Two others MOVs 5 and 7 reacted with both. Using the peroxidase-anti- peroxidase (PAP) histochemical method, the target epitopes of MOV 1 were located in the cuticle's basal and cortical layers, those of MOV 2 in the cortical layer; whilst MOV 3-7 stained the basal layer. A sandwich ELISA was then developed. The trapping polyclonal antibody was raised in rabbits utilising the same antigens as for preparation of the MOVs. Once captured on microtiter plates, target antigens were identified by the sequential binding of a MOV, followed by a goat anti-mouse globulin/peroxidase or alkaline phosphatase conjugate that catalysed a colorimetric reaction in the presence of appropriate substrates. In this system, MOV 1 emerged as the most specific and potent reagent capable of recognizing antigens of Onchocerca sp. with a minimal detection limit of 78 ng per test. MOV 1, failed to react with extracts of Loa Loa, Ascaris lumbricoides and Ascaris suum in the test. The developed assay relied on the use of MOV 1, required only 1 ml of urine or 0.05 ml serum. About 97.8% of the 47 urines and 50% of the 20 sera from patients studied gave positive results. Only 1 (3%) of 32 control urines and up to 80% of the 10 control sera studied tested positive, suggesting urine as a better specimen source.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-mediated and monoclonal antibody-dependent killing of Onchocerca volvulus microfilariae.

To identify the target antigens implicated in the adherence and killing of microfilariae (mf) by leucocytes, we incubated nodular mf and leucocytes in the presence of anti-cuticular monoclonal antibodies (MOVs) and fresh serum. Leucocyte donors were patients with a mean age of 37 years (with 0-1 mf/snip), who had lived in the endemic village studied for at least 10 years. After 16-20 h of incubation, up to 74% of the mf could be seen with 10 or more cells adhering to them. By 36-40 h up to 54% of the mf had been killed by the leucocytes in the presence of a cocktail of monoclonal antibodies termed MOV GIV. The degree of killing in control experiments with the monoclonal antibody MOV 1 remained lower (P less than 0.05), ranging from 0.0 to 4.5% of mf with initial viability of 90-95%. Western blotting revealed MOV GIV prominent target antigens of 10.5, 18.0, 23.5 and 27 kDa in crude surface extracts of female O. volvulus. The detected antigens may play a role in host protection.

Identification of the surface polypeptide antigens of the infective larvae of Onchocerca volvulus.

The surface polypeptide antigens of third-stage infective larvae (L3) and adult males of Onchocerca volvulus have been compared after iodogen-catalysed radioiodination, separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in beta-mercaptoethanol followed by autoradiography. L3 surfaces contained polypeptides with apparent molecular weights of 14, 18.5, 26, 34, 51, 68 and 130 kDa, whereas the adults contained 14, 19, 26, 34, 37.5 and 68 kDa components. By immunoprecipitation with patient's sera, only the 14 and 18.5 kDa components were shown to be antigenic on the L3, the other components being unreactive with patients' antibodies. Under similar conditions, 4 of the 6 adult male polypeptides reacted with patients' antisera, confirming their antigenic nature. Lentil lectin and immunosorbent chromatography of the surface components revealed the 18.5 kDa and 68 kDa antigens of L3 and adult males respectively to be glycoproteins. The apparently weak reactivity of L3 surface components with host antibody may be part of an escape mechanism that favours the establishment of O. volvulus in human hosts.

Identification of different radiolabelled antigens of the developmental stages of Onchocerca volvulus.

By using radioiodination methods which are thought to label preferentially the surface followed by SDS-PAGE and autoradiography, components of different developmental stages of O. volvulus have been identified. Between 2 and 10 polypeptide antigens were revealed on infective larvae (L3), females, males, eggs, nodular and skin microfilariae by using immunoblotting assays with human onchocerciasis sera. Antigen recognition did not vary with the density of skin microfilariae in the patients from whom the sera were obtained. Some of the antigens seemed to be stage specific; for example, antigens of 31 kDa which were detected only on skin microfilariae, or the 67.5 and 25 kDa components that occurred on the adult females, but were absent from adult males. Some of these antigens were also identified as glycoproteins. A 68 kDa glycoprotein was found in adult females, males and nodular microfilariae. Two glycoproteins of 74 and 45 kDa were found on egg shells, and a 18.5 kDa glycoprotein was recovered from L3. Type VI collagen was found with a specific antiserum on skin microfilariae, but not on eggs and females. Laminin was found on nodular mf. It is concluded that the changing antigenic profiles of the worm stages and the coating of these worms with connective tissue epitopes contribute to the evasion of host immunity.

Isolation and biochemical composition of the cuticle of Onchocerca volvulus.

A preparation of the cuticle of Onchocerca volvulus was obtained by extracting worm fragments in an series of buffers with 1.5% Triton-X-100 and 3% Sodium dodecyl sulfate (SDS). Electron micrographs of worm fragments, treated with the detergents or collagenase showed that our methods had been effective in isolating the cuticle from the other organs of the parasite. The cuticular preparation was found to contain 19 different amino acids with glycine (23.4%); proline (11.23%); hydroxyproline (10%); and glutamic acid (9.4%) being the most abundant. Hydroxylysine was present in small amounts (0.04%). Total reducing sugar was determined to be 5.3 mg per gram dry weight of the preparation. The cuticular preparation was solubilized by boiling in 2-mercaptoethanol and shown by SDS-PAGE to contain at least 10 different polypeptides in the Mr range 17,000-163,000. Five of these polypeptides with apparent Mr respectively of 33,000; 67,000; 74,000, 88,500 and 114,000 were isolated by preparative gel electrophoresis and their amino acid compositions shown to be similar to that of invertebrate collagens. We conclude that the cuticle of O. volvulus contains collagen-like proteins held together by disulfide bridges.