Wayne M. Meyers

Insects in the transmission of Mycobacterium ulcerans infection

Buruli ulcer, caused by Mycobacterium ulcerans, is a common disease of skin and bones, which usually occurs in the vicinity of rural tropical wetlands. Epidemiological studies have not established a natural reservoir or mode of transmission. We collected 30 plants from swamps in areas of Benin (Ouinhi and Zangnanado) and Ghana (Tontokrom) endemic for Buruli ulcer. We tested roots, stems, and leaves for M ulcerans by culture and nested PCR. , 3 We also studied five water bugs found on the roots of three of these plants. Of the 95 samples analysed, only the five insects were positive by PCR for M ulcerans. These insects were from the roots of plants belonging to the C y p e r u s, P a n i c u m, and E i c h h o r n i a genera. Two of them from the roots of the C y p e r u s a n d P a n i c u m were not identified but were probably water bugs of the family Naucoridae. Roots of E i c h h o r n i a sheltered three aquatic bugs, one belonging to the genus N a u c o r i s ( f a m i l y Naucoridae) and two to the genus D i p l o n y c h u s ( f a m i l y Belostomatidae). Only the insects were positive; not the roots, stems, or leaves. Three different genera of plants are involved, suggesting that the type of plant is not a factor, but it is the aquatic bugs in the roots that are of interest. These bugs are aggressive predators of other species of aquatic arthropods and molluscs. They fly from one nearby swamp or pond to another, and may bite human beings. Water bugs belonging to the generae N a u c o r i s and Diplonychus often bite villagers. A study of ten water bugs from the same areas showed that two were positive for acid-fast bacilli. Only occasional acid-fast bacilli were seen, suggesting that water bugs were mechanical vectors of M ulcerans. We propose a new hypothesis for a source of M ulcerans a n d one mode of its transmission to human beings. M ulcerans survives best under low oxygen tensions, such as exist in mud in the bottom of swamps. Some water-filtering organisms could concentrate M ulcerans and be ingested by waterdwelling predators, which are then a passive reservoir for M ulcerans. Bites of these insects or contamination of human skin by their faeces may explain why direct contact with water is not necessary to acquire Buruli ulcer. Trauma seems essential for the introduction of M ulcerans into the skin. T h e aetiological agent may be introduced directly by bites of these insects, or by trauma at skin sites contaminated by insect products containing M ulcerans. There may also be parallel modes of transmission of M ulcerans, such as by aerosols from the surface of swamps. If confirmed, we believe this would be the first implication of insects in the transmission of a mycobacterial disease.

Fatal strongyloidiasis in immunosuppressed patients

Abstract Cell-mediated immunity is a major bulwark of defense against certain viruses, intracellular bacteria, fungi and protozoa. Cellmediated immunity against helminthic infections is inadequately understood. Thirty-two patients with strongyloidiasis who were studied at autopsy had underlying diseases characterized by depressed cell-mediated immunity, including lepromatous leprosy, malignant tumors, protein-calorie malnutrition, burn, radiation, advanced tuberculosis, tertiary syphilis or pancytopenia. At autopsy defective cell-mediated immunity against strongyloides was manifested by a lack of a granulomatous immune response to larvae in the tissues. Depletion of lymphocytes in thymus glands and in the thymus-dependent areas of lymph nodes and spleens in the hosts indicated in inadequate capacity of cell-mediated immunity for preventing Strongyloides stercoralis.

First Cultivation and Characterization of Mycobacterium ulcerans from the Environment

Background Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin. Methodology/Principal Findings One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5′ end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3′ end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone. Conclusion Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.

Mycobacterium ulcerans Disease (Buruli Ulcer) in Rural Hospital, Southern Benin, 1997-2001

Hospital data show that Buruli ulcer is highly endemic in southern Benin.

Mycobacterium bovis BCG Vaccination as Prophylaxis against Mycobacterium ulcerans Osteomyelitis in Buruli Ulcer Disease

ABSTRACT Mycobacterium ulcerans disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. Clinically, the disease presents in the skin as either nonulcerative or ulcerative forms and often invades bones either subjacent to the skin lesion (contiguous osteomyelitis) or remote from the skin lesion (metastatic osteomyelitis). Osteomyelitis represents a severe form of the disease that often requires numerous surgical interventions, even amputations. Surgery is accepted as the present definitive treatment for BU. In the absence of an effective drug treatment, the need for the development of preventive and control strategies becomes paramount. No specific vaccine, however, is presently available for BU. Of 372 consecutive patients in Benin presenting with BU (confirmed by microbiological and histopathological analyses) whose Mycobacterium bovis BCG scar statuses were known, 196 children (<15 years old) and 108 adults had neonatal BCG vaccination scars. Of 196 children with BCG scars, 17 (8.7%) had osteomyelitis, while 7 of 28 children without BCG scars (25.0%) had osteomyelitis. Of 108 adults with BCG scars, 17 (15.7%) had osteomyelitis, while 14 of 40 adults without BCG scars (35.0%) had osteomyelitis. Our results show that effective BCG vaccination at birth provides significant protection against the development of M. ulcerans osteomyelitis in children and adults. Therefore, health authorities should give attention to the enhancement of neonatal BCG vaccination coverage in all countries of Africa where BU is endemic. Protection against severe forms of BU and childhood tuberculosis would likewise be improved by this intervention.

Mycobacterium ulcerans toxic macrolide, mycolactone modulates the host immune response and cellular location of M. ulcerans in vitro and in vivo

Mycobacterium ulcerans produces an extracellular cutaneous infection (Buruli ulcer) characterized by immunosuppression. This is in stark contrast to all other pathogenic Mycobacteria species that cause intracellular, granulomatous infections. The unique mycobacterial pathology of M. ulcerans infection is attributed to a plasmid‐encoded immunomodulatory macrolide toxin, mycolactone. In this article we explore the role of mycolactone in the virulence of M. ulcerans using mycolactone and genetically defined mycolactone negative mutants. In a guinea pig infection model wild‐type (WT) M. ulcerans produces an extracellular infection whereas mycolactone negative mutants produce an intracellular inflammatory infection similar to that of Mycobacterium marinum. Although mycolactone negative mutants are avirulent, they persist for at least 6 weeks. Chemical complementation of M. ulcerans mutants with mycolactone restores WT M. ulcerans pathology. Mycolactone negative mutants are capable of growth within macrophages in vitro whereas macrophages are killed by WT M. ulcerans. The ability of mycolactone to caused delayed cell death via apoptosis has been reported. However, mycolactone also causes cell death via necrosis. In vitro mycolactone has antiphagocytic properties. Neither WT M. ulcerans nor mycolactone negative strains are strong neutrophil attractants. These results suggest that mycolactone is largely responsible for the unique pathology produced by M. ulcerans.

Potential Role for Fish in Transmission of Mycobacterium ulcerans Disease (Buruli Ulcer): an Environmental Study

ABSTRACT This study reports a potential role that fish may play in the transmission of Mycobacterium ulcerans disease (Buruli ulcer). Fish found positive for M. ulcerans DNA all appear to feed on insects or plankton and are believed to concentrate M. ulcerans from this usual food source. These observations provide additional data supporting our previous hypothesis on sources of M. ulcerans and modes of transmission.

Risk Factors for Buruli Ulcer, Benin

Disease was associated with age, place of residence, and water sources in all age groups.

Mycobacterium ulcerans disease: role of age and gender in incidence and morbidity

During the 5‐year period, 1997–2001, 1700 patients with a clinical diagnosis of Mycobacterium ulcerans disease [Buruli ulcer (BU)] were treated at the Centre Sanitaire et Nutritionnel Gbemoten, Zagnanado, Benin. The patients lived in the four regions of southern Benin: Atlantique, Mono, Oueme and Zou, with the largest number coming from the Zou Region where the centre is located. The median age of BU patients was 15 years (q1 = 7, q3 = 30). Lower limbs are involved 3.2 times more frequently than upper limbs in older patients and younger patients have the highest prevalence of multiple lesions. The latter are frequently associated with bone lesions. Specific detection rates for age and gender showed a distribution with maximum peaks in the 10–14 years group and among adults between 75 and 79 years. Over 59 years, males are more at risk of developing M. ulcerans disease than females. Children under 15 years represent the largest part of the BU disease burden and of the general population. The highest detection rates (per 100 000 population) were in the 75–79‐year‐old patients. The most likely explanation of this was reactivation of disease from a latent infection of M. ulcerans. Educational programmes should target especially these two groups of population at risk.

Evidence for an Intramacrophage Growth Phase of Mycobacterium ulcerans

ABSTRACT Mycobacterium ulcerans is the etiologic agent of Buruli ulcer (BU), an emerging tropical skin disease. Virulent M. ulcerans secretes mycolactone, a cytotoxic exotoxin with a key pathogenic role. M. ulcerans in biopsy specimens has been described as an extracellular bacillus. In vitro assays have suggested a mycolactone-induced inhibition of M. ulcerans uptake by macrophages in which its proliferation has not been demonstrated. Therefore, and uniquely for a mycobacterium, M. ulcerans has been classified as an extracellular pathogen. In specimens from patients and in mouse footpad lesions, extracellular bacilli were concentrated in central necrotic acellular areas; however, we found bacilli within macrophages in surrounding inflammatory infiltrates. We demonstrated that mycolactone-producing M. ulcerans isolates are efficiently phagocytosed by murine macrophages, indicating that the extracellular location of M. ulcerans is not a result of inhibition of phagocytosis. Additionally, we found that M. ulcerans multiplies inside cultured mouse macrophages when low multiplicities of infection are used to prevent early mycolactone-associated cytotoxicity. Following the proliferation phase within macrophages, M. ulcerans induces the lysis of the infected host cells, becoming extracellular. Our data show that M. ulcerans, like M. tuberculosis, is an intracellular parasite with phases of intramacrophage and extracellular multiplication. The occurrence of an intramacrophage phase is in accordance with the development of cell-mediated and delayed-type hypersensitivity responses in BU patients.