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.

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 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.

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.