Investigation of human haemotropic Mycoplasma infections using a novel generic haemoplasma qPCR assay on blood samples and blood smears.

The aim of this study was to develop quantitative real-time (q)PCR assays to detect all known haemoplasma species, and a human housekeeping gene in order to demonstrate both successful DNA extraction from clinical samples and to test for sample inhibition, and to apply these qPCRs to human blood samples and blood smears. Sensitive and specific generic haemoplasma qPCR assays were developed to amplify haemoplasma species, as well as human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal amplification control. An optimized technique for extracting DNA from stained blood smears was also developed. These methods were applied to anonymized blood samples obtained from 100 human immunodeficiency virus (HIV)-infected South Africans and 920 UK patients undergoing haematological examination, and to 15 blood smears recruited from previous studies describing human haemoplasmosis. Human GAPDH levels were acceptable in all but three of the blood samples and all but two of the blood smears. The latter could have arisen due to DNA degradation due to the old age (over 35 years) of these smears. Haemoplasma infection was found in one HIV-infected South African, but the species could not be characterized due to the very low levels of DNA present. This report describes novel extraction and qPCR methodologies for haemoplasma screening. Previously reported human haemoplasmosis based on cytological diagnosis alone should be viewed with caution.

"Nanobacterium sanguineum"--is it a new life-form in search of human ailment or commensal: overview of its transmissibility and chemical means of intervention.

Morphological, cultural, and immuno-histochemical characteristics of "Nanobacterium sanguineum" (NB) described in the literature are reviewed. NB is reported to be a motile, Gram negative organism that divides by binary fission within a calcium-coated slimy shell; this yeast-like shell replicates by budding. It measures between 20 and 200 nm with a unique structure containing 16S ribosomal RNA. NB has been observed by electron microscopy in coronary artery plaques (CAD) and in kidney stones (KS) found in renal diseases. On the basis of supportive literature, we suggest that NB is not only present in the human body but also has auxiliary association with human ailments without a specific etiological role; anti-NB antibody has been detected in subjects with calcified lesions and inflammation in diverse ailments including choriodecidual inflammation in pregnancy, ovarian cancers, arthritis and even Alzheimer's disease. More recent report on the detection and vertical transmission of NB antigen and anti-NB antibody in HIV-infected mothers supports the view that NB might be an important opportunistic infective agent contributing to HIV pathology; we note that the presence of viable and transmitting NB was not studied and suggest further studies to establish vertical transmission of NB in HIV-infected persons. On the basis of the foregoing we suggest that NB possibly exacerbates human ailments and raise the question: Is NB a new life-form in search of human ailment or a commensal organism? Recognizing the presence of NB in the human body, we discuss clinical trials, reported in the literature relevant to its eradication, with a rectal suppository containing very high amounts of disodium EDTA and tetracycline. We suggest that tetracycline in this formulation acted in combination with EDTA, more as a chelating agent than an antibiotic; oxytetracycline- a non-chelating form of tetracycline-does not inhibit or kill NB. Evaluation of anti-NB effect of orally administrable and potentially safer as well as therapeutically more acceptable chelating agent -ascorbic acid, acting alone or in combination with antibiotics-that eradicates another slime forming bacterium - Pseudomonas aeruginosa - in children with cystic fibrosis, is suggested.

Repellent efficacy of four commercial DEET lotions against Amblyomma hebraeum (Acari: Ixodidae), the principal vector of Rickettsia africae in southern Africa.

African tick bite fever, caused by Rickettsia africae, is an emerging zoonotic infection in rural sub-Saharan Africa and the French West Indies. We tested the repellent efficacy of four commercial diethyl-3-methylbenzamide (DEET) lotions against Amblyomma hebraeum Koch, the principal vector of R. africae in southern Africa, by using a human bioassay in which repellent-treated fingers were presented to questing tick nymphs hourly for 4h. Three lotions with 19.5, 31.6 and 80% DEET concentrations, respectively, had a repellent efficacy of > or =90% at 1h post-application, of > or =77% at 2h post-application and of <70% during the rest of the experiment. By contrast, a lotion with 2% DEET plus 1% citronella oil provided only a 59% repellency at 1h post-application, with a drop to <22% during the following hours. Hourly negative control trials repelled <5% of tick attacks. Our results suggest that commercial repellents containing > or =19.5% DEET provide a significant but short-lasting protection against questing A. hebraeum ticks.

Diversity of bartonellae associated with small mammals inhabiting Free State province, South Africa.

The prevalence and diversity of bartonellae infecting the blood of 10 small mammal species inhabiting nine Nature Reserves of the Free State province, South Africa, was assessed using phenotypic, genotypic and phylogenetic methods. Of 86 small mammals sampled, 38 animals belonging to five different species yielded putative bartonellae. Thirty-two isolates were confirmed as bartonellae and were characterized by comparison of partial citrate synthase gene (gltA) sequences. Phylogenetic reconstructions derived from alignment of these sequences with those available for other bartonellae indicated that the South African rodent-associated isolates formed two distinct clades within the radius of the genus Bartonella. One of these clades also included recognized Bartonella species associated with rodents native to Eurasia but not to the New World, whereas the second clade contained exclusively isolates associated with South African rodents. Comparison of gltA sequences delineated the isolates into a number of ecologically distinct populations and provided an indication that a combination of phylogenetics and the identification of sequence clusters in housekeeping protein-encoding genes could be developed as a key criterion in the classification of bartonellae. This study is the first to investigate wildlife-associated bartonellae in Africa, adding support to their ubiquity and broad diversity and to the paradigm that the phylogenetic positions of the Bartonella species encountered today have been influenced by the geographical distribution of their hosts.

Rickettsia mongolotimonae infection in South Africa.

We report the first laboratory-confirmed case of Rickettsia mongolotimonae infection in Africa. The patient sought treatment for an eschar on his toe; lymphangitis, severe headaches, and fever subsequently developed. After a regimen of doxycycline, symptoms rapidly resolved. R. mongolotimonae infection was diagnosed retrospectively by serologic tests and molecular-based detection of the organism in biopsy specimens of eschar material.

Update on spotted fever group Rickettsiae in South Africa.

Until very recently, Mediterranean spotted fever caused by Rickettsia conorii was the only spotted fever group (SFG) rickettsioses recognized in southern Africa. However, increasing medical awareness of tick-borne infections, together with the introduction of improved isolation methods and the availability of molecular techniques, have led to the identification of several new SFG rickettsioses in the region. African tick bite fever, caused by Rickettsia africae, is currently the most important of these new rickettsioses, affecting large numbers of international travellers each year, but infections due to Rickettsia aeschlimannii and Rickettsia mongolotimonae have also been recently encountered. In this review, we describe the current status of the epidemiology, microbiology, clinical presentation, diagnosis, treatment, and prevention of SFG rickettsioses in southern Africa.

Rickettsia mongolotimonae Infection in South Africa

We report the first laboratory-confirmed case of Rickettsia mongolotimonae infection in Africa. The patient sought treatment for an eschar on his toe; lymphangitis; severe headaches; and fever subsequently developed. After a regimen of doxycycline; symptoms rapidly resolved. R. mongolotimonae infection was diagnosed retrospectively by serologic tests and molecular-based detection of the organism in biopsy specimens of eschar material

A preliminary investigation into light-modulated replication of nanobacteria and heart disease.

OBJECTIVE: The purpose of this preliminary study is to evaluate the effect of various wavelengths of light on nanobacteria (NB). BACKGROUND DATA: NB and mitochondria use light for biological processes. NB have been described as multifunctional primordial nanovesicles with the potential to utilize solar energy for replication. NB produce slime, a process common to living bacteria. Slime release is an evolutionary important stress-dependent phenomenon increasing the survival chance of individual bacteria in a colony. In the cardiovascular system, stress-induced bacterial colony formation may lead to a deposition of plaque. METHODS: Cultured NB were irradiated with NASA-LEDs at different wavelengths of light: 670, 728 and 880 nm. Light intensities were about 500k Wm(-2), and energy density was 1 x 10(4) J m(-2). RESULTS: Monochromatic light clearly affected replication of NB. Maximum replication was achieved at 670 nm. CONCLUSIONS: The results indicate that suitable wavelengths of light could be instrumental in elevating the vitality level of NB, preventing the production of NB-mediated slime, and simultaneously increasing the vitality level of mitochondria. The finding could stimulate the design of cooperative therapy concepts that could reduce death caused by myocardial infarcts.

Repellent efficacy of DEET and KBR 3023 against Amblyomma hebraeum (Acari: Ixodidae).

The bont tick, Amblyomma hebraeum Koch, is the principal vector of Rickettsia africae, the agent of African tick bite fever, in southern Africa. In contrast to most other hard ticks, members of the genus Amblyomma exhibit a characteristic attack host-seeking behavior. The efficacy of topically applied 20% lotions of DEET (diethyl-3-methylbenzamide), the currently considered reference repellent substance, and KBR 3023, a recently developed piperidine compound, was evaluated against laboratory-reared A. hebraeum nymphs on treated fingers. Both substances repelled >85% of nymph attacks at 0 and 1 h postapplication. At 2, 3, and 4 h, the repellent efficacies of DEET were 84%, 68% and 71%, whereas those of KBR 3023 were 56, 55, and 54%. The observed differences between the two test substances were statistically significant only at 2 h postapplication. This first study of topical repellents against A. hebraeum suggests that 20% lotions of DEET and KBR 3023 are effective for 2 and 1 h, respectively, but that the repellent efficacies decrease thereafter.