Risk Factors and Associated Outcomes of Virulence Genes eae, entB, and pipD Carriage in Escherichia coli, Klebsiella pneumoniae, and Salmonella spp. From HIV-1 and HIV-Negative Gastroenteritis Patients in the Dschang Regional Hospital Annex.

Background Enterobacteriaceae is one of the main families of gram-negative bacilli responsible for serious infections in humans. The severity of infection by these bacteria is a product of many factors, including virulence properties and antimicrobial resistance. This severity may be further intensified if there is an association between these factors and a depressed immune system, such as in HIV patients. This study aimed to determine the distribution of representative virulence genes among key Enterobacteriaceae isolates from HIV-1 and non-HIV gastroenteritis patients and the relationship between carrying these virulence genes and antimicrobial susceptibility, seropositive status, and severity of symptoms associated with Enterobacteriaceae infections in Dschang Regional Hospital Annex. Methodology A total of 200 gastroenteritis patients (100 HIV-1 and 100 non-HIV patients) were selected and evaluated for symptoms associated with gastroenteritis. Stool samples were obtained and cultured, from which Escherichia coli, Klebsiella pneumoniae, and Salmonella spp. isolates were obtained. Antibiotic susceptibility tests were performed on the isolates by agar disc diffusion using commonly used antibiotics. These isolates were tested for the possession of virulence genes by polymerase chain reaction (PCR); eae for E. coli, entB for K. pneumoniae, and pipD for Salmonella spp. Correlation tests and risk assessments were performed between the presence of virulence genes, antibiotic resistance, and specific symptoms. Results The isolates obtained from HIV-positive and HIV-negative patients were, respectively, 61 against 62 for E. coli, 10 against 21 for K. pneumoniae, and 11 against 15 for Salmonella spp.These organisms showed the highest resistance to amoxicillin and clavulanic acid, while the least resistance was observed against ofloxacin, gentamicin, and amikacin in both groups of patients. The virulence genes showed a generally higher occurrence in isolates from HIV-negative patients than HIV-positive patients, with the eae gene 5/61 (8.20%) against 12/62 (19.35%), the entB gene 4/10 (40.00%) against 14/21 (66.66%), and the pipD gene 5/11 (45.45%) against 7/15 (46.46%) in HIV-positive and negative patients, respectively. There was a significant correlation between eae gene carriage and resistance against imipenem (p = 0.047), gentamycin (p = 0.047), and doxycycline (p = 0.029); entB gene carriage and resistance toward levofloxacin (p = 0.017) in K. pneumoniae; and pipD gene carriage and resistance against levofloxacin (p = 0.039), imipenem (p = 0.041), and doxycycline (p = 0.042). The carriage of the virulence genes was seen to be a stronger risk only for the resistance of K. pneumoniae to ceftriaxone (odds ratio (OR) = 2.286) and gentamycin (OR = 3.000), and Salmonella spp. against imipenem (OR = 2.750) and doxycycline (OR = 2.118). The development of severe symptoms correlated significantly with virulence gene carriage in isolates, mainly in HIV-positive patients with eae (p = 0.017) and pipD (p = 0.025), with a strong risk association with the pipD gene (OR = 2.665). Conclusions Antibiotic resistance was associated with virulence gene carriage, indicating that virulence and antibiotic resistance can associate their effects and contribute to poor outcomes in the treatment of bacterial diseases in HIV patients. The possession of virulence genes increased the severity of symptoms associated with gastroenteritis in HIV-positive patients.

Isolation of a 14 kDa antigen from Taenia solium cyst fluid by HPLC and its evaluation in enzyme linked immunosorbent assay for diagnosis of porcine cysticercosis.

A fraction with a major band of 14kDa was obtained from crude cyst fluid of Taenia solium cysticerci by 2-step chromatography. A first fraction isolated by gel filtration (Sephacryl S-300 high resolution) was purified using an anion exchange column (Mono Q HR 5/5) on high performance liquid chromatography. Evaluation of the analytic sensitivity of this fraction (F3) was carried out in an antibody enzyme linked immunosorbent assay (Ab-ELISA-F3) using serum samples from pigs experimentally infected with different doses of T. solium eggs. The cross-reactivity of F3 was evaluated with serum samples from pigs that were naturally or experimentally infected with Taenia hydatigena, Taenia saginata asiatica, Fasciola hepatica, Trichinella spiralis, Metastrongylus apri, Trypanosoma congolense and Sarcoptes scabiei, and with serum samples of rabbits hyper-immunised with metacestode cyst fluid of T. hydatigena and T. solium. Antibody titres of lightly or heavily infected pigs differed in their kinetics. However, the increase in F3-specific antibodies could not be related to the infection level. Analysis of the specificity of the F3 showed that serum samples of pigs infected with other parasites did not recognise this antigen. Cross-reaction with T. hydatigena occurred in ELISA using cyst fluid as antigen, but the F3 antigen fraction was not recognized by rabbit hyper-immune serum samples to T. hydatigena. Evaluation of the diagnostic sensitivity and specificity of the Ab-ELISA-F3 was done by a non-parametric receiver operating characteristic (ROC) analysis using 66 serum samples from Zambian village pigs. The total number of cysticerci of these pigs was determined by dissection (28 pigs harboured T. solium cysticerci and 38 were negative at dissection). In addition, 58 serum samples from Cameroonian pigs (28 pigs from cysticercosis-free farms and 30 pigs with cysticerci at tongue inspection) were used in a separate ROC analysis. The results from the ROC analysis yielded a low diagnostic value (area under ROC curve=0.48) with the sera from the Zambian pigs while a relatively high diagnostic value was obtained with the sera from Cameroonian pigs (area under ROC curve=0.78). The main factor contributing to a low diagnostic value based on the Zambian serum samples seemed to be the false-positive reactions that were likely caused by the occurrence of transient antibodies in the non-infected animals.

[Development of an antigen detection dot blot assay for the diagnosis of human onchocerciasis based on the biotin-avidin binding system]. / Développement d'un test dot blot de détection d'antigène, basé sur le système de fixation biotine- avidine, pour le diagnostic de l'onchocercose humaine.

The development of a highly sensitive and specific diagnostic test is of urgent need for the field assessment of human onchocerciasis and for monitoring the success of control programs. We report here the development and evaluation of a Dot blot Immunobinding Assay (DIA-BA) based on the biotin-avidin binding system, for the detection of O. volvulus specific antigens in body fluids. Specific antibodies were produced by immunizing rabbits with the O. volvulus recombinant antigen Oncho-C71 and labelled with biotin. The biotinylated probes were then used to detect O. volvulus specific antigens initially blotted onto a nitrocellulose membrane. The smallest amount of blotted antigens detectable by the new test is 0.5ng, 1ng, 1ng and 2ng respectively in urine, dermal fluid, tears and serum samples. Out of 456 onchocerciasis endemic subjects examined, 98.4%, 96.5%, 90.8% and 75.0% were positive by the DIA-BA test on urine, dermal fluid, tears and serum respectively The test was most sensitive (100%) when used on urine and least (54.76%) when used on serum from skin snip positive subjects. The specificity of the test, determined amongst non-exposed individuals, was 100% on all but for dermal fluid samples (97.5%). Also, the color intensities on the blot were observed to positively correlate (r = 0.8 on urine) with the skin microfilaria loads on the individuals. We conclude that DIA-BA test could be very useful for mass diagnosis of prepatent, of low and high level infections due to O. volvulus.

Development and evaluation of an antigen detection dipstick assay for the diagnosis of human onchocerciasis.

To improve on the diagnosis of onchocerciasis, especially light infections, we developed and evaluated an oncho-dipstick test based on the detection of Onchocerca volvulus specific antigens in urine and tears. The test was able to detect as little as 25 ng/ml of parasite specific antigens in samples and took as little as 3 h. Evaluation of the assay on 456 residents of an onchocerciasis hyperendermic area in Cameroon resulted in 408 (89.5%) positives in urine and 374 (82%) positives in tears. The prevalence of onchocerciasis in the study area, as determined by Rapid Epidemiological Mapping of Onchocerciasis (REMO) and skin snip methods, was 52 and 36.8%, respectively. The sensitivity of the oncho-dipstick assay was 100% in urine and 92% in tears; its specificity was 100% in both. Concordance between urine and tear test results from the same individuals was 87%. The test strips were sufficiently reactive when left at room temperature for up to 8 months. The test would be useful for laboratory diagnosis of onchocerciasis in low transmission zones and to ascertain successful treatment of patients in experimental drug studies.

[Proteolytic activity of adult worm extracts of Onchocerca volvulus]. / Activité protéolytique de l'extrait de vers adultes de l'Onchocerca volvulus.

Studying proteolytic activity of Onchocerca volvulus (nematode causing "river blindness") shows that it is able to digest a variety of substrates such as: azoalbumine, azocoll and elastin-orcein with specific activity of 0.28, 0.57 and 1.48 mg/hour/mg of extract respectively. These enzymes are active at various pH such as pH 5.0, 8.0 and 10.0 with highest activity at pH 8.0. The effect of specific inhibitors and activators indicates that the extract might contain serine, metallo and thyoproteases. The electrophoresis of the extract on a polyacrylamide gel copolymerized with gelatin shows many proteins with enzymatic activities with molecular weight of 16.6, 43.6, 45.7, 56.2, 60.2, 61.6 and 63.1 KD respectively. The Onchocerca volvulus worm contains proteases of various enzymatic activities: a non specific activity on protein such as on azoalbumin and specific activities on collagen and elastin. These enzymes could play an important role in the survival of parasites in human hosts.

Novel, sensitive and low-cost diagnostic tests for 'river blindness'--detection of specific antigens in tears, urine and dermal fluid.

Sensitive, specific and low-cost diagnostic tests for onchocerciasis are indispensable for monitoring the efficacy of control programs, as well as for preventing blindness (when the tests are combined with efficacious chemotherapy. Three new tests to detect Onchocerca-specific antigens in tears, dermal fluid and urine employ antibodies to O. volvulus-specific recombinant proteins, Oncho-C27 and OvD3B, encoded by genes within the immunodominant Onchocerca OV 33-3 gene family, and expressed in yeast and in E. coli, respectively. In these assays, Onchocerca-specific antigens in test samples are bound onto a solid surface and revealed using appropriate enzyme-labelled antibodies. Proteins in the samples are first transferred to Hybond-N + membrane disks or nitrocellulose paper using either a transblot or a dotblot machine, and then reacted with specific O. volvulus antibodies. Bound antibodies are revealed with species-specific peroxidase-labelled antibodies and peroxidase substrate. Positive tests give a brown colour. In one of the two assays developed to detect Onchocerca antigens in tears, the sensitivity was enhanced by first adsorbing the specific antibodies onto the membrane surface in order to immobilize and concentrate the Onchocerca-specific antigen molecules on the membrane. The specificity of the recombinant proteins for Onchocerca volvulus had been verified by ELISA, classical Western blot and modified DSIA. The tests are a dipstick immunobinding assay for ocular microfilariae (DSIA), a transblot immunobinding assay for the detection of skin microfilariae (TADA) and a dot-blot immunobinding assay for detecting urinary microfilariae and their antigens (DIA). Their specificity and sensitivity were evaluated in the field on 110 subjects with proven ocular microfilariae, 130 subjects with clinical and parasitological evidence of onchocerciasis, 25 subjects infected with other helminths and 120 normal controls. The minimal detection limits of Oncho-C27 protein by DSIA, TADA and DIA were 500 ng/ml, 154 ng/ml and 508 ng/ml, respectively By contrast, their sensitivities were: 100% for DSIA and 82.5% for TADA employed on samples of tears; 97% for TADA skin test and 96% for DIA used on urine samples.

Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast.

A yeast (Saccharomyces cerevisiae) expression system has been adapted to produce reagent quantities of a major Onchocerca antigen, Ov33. Using a pool of monoclonal antibodies produced against third-stage larvae, a cDNA library constructed from adult O. volvulus worms was screened. Twenty-seven cDNAs were isolated, two of which had sequence homology to Ov33, a putative aspartyl protease inhibitor, which is the immunodominant antigen of O. volvulus. These cDNAs were expressed at high levels intracellularly or through the secretory pathway of S. cerevisiae. Localization studies using antisera produced against purified recombinant protein demonstrated that Ov33 is a very abundant parasite protein present in the hypodermis, muscle, and uterus of female worms, as well as in embryonic microfilariae. The soluble recombinant protein secreted by yeast (C71) demonstrated inhibitory activity against the aspartyl protease pepsin. Antibodies to the recombinant protein-mediated leukocyte adherence to and killing of skin microfilariae. The sensitivity of a diagnostic test using recombinant Ov33 was evaluated using sera from 441 patients. The mean sensitivities for the two recombinant constructs, C27 and C71, were 82.2% and 85.4%, respectively. The combined sensitivity using both recombinant proteins was 94%.

Onchocerca volvulus: identification and characterization of an immunogenic eggshell protein (Oveg1).

A major antigen recognized by human sera in Onchocerca volvulus infections is a parasite eggshell protein. The cDNA clone for this antigen was isolated from a lambda gt11 O. volvulus cDNA library using antisera from patients with high microfilarial counts. Sequence analysis of the cDNA clone predicts a polyglutamine repeat near the 5' end of the cDNA, and a motif of four arginines near the 3' end, reminiscent of that found in many regulatory proteins. The cDNA was subcloned into a yeast expression vector and reagent quantities of recombinant antigen produced in Saccharomyces cerevisiae. Antisera produced to the recombinant purified protein localized the antigen to the eggshell of developing microfilariae within the adult female uterus. No other sites of Oveg1 expression were noted in adult worms, but labeling was seen in internal membrane structures of L3 larvae. Sera from infected chimps recognized Oveg1 only after infections became patent. Sera from infected humans showed reactivity to Oveg1 that varied from 39 to 95%, depending upon the geographic location.

Comparative studies of clinical groups of patients in an onchocerciasis endemic area for evidence of immune-mediated protection.

Four clinical groups of persons from an area endemic for onchocerciasis were compared using certain immunological parameters. The groups were: generalised onchocerciasis, patients with restricted distribution of onchocercal skin lesions, microfilaredermia patients with no clinical manifestations, and a group which clinically, had successfully resisted the infection. Specific serum antibodies to O. volvulus antigens were found in all groups. The IgG specific antibodies were highest in patients with generalised onchocerciasis and lowest in the group who had apparently contained the infection. The sera of persons from the latter group mediated leukocyte adherence to and immobilised microfilariae of O. volvulus. Using the Western blot technique, there were no onchocercal protein antigens that reacted exclusively with sera from the "protected group". However, the staining of the reaction bands was most intense when sera from this patient group reacted with low molecular weight specific onchocercal antigens (M. W. 10-57 KD).