P. W. M. Hermans

A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti : Characterization of an exceptional isolate from Africa

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated M. tuberculosis.

Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment‐length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30‐bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).

Treponema pallidum subspecies pallidum (Nichols) and Treponema pallidum subspecies pertenue (CDC 2575) differ in at least one nucleotide: comparison of two homologous antigens.

In an attempt to identify antigenic differences between Treponema pallidum subsp. pallidum (T. pallidum) and Treponema pallidum subsp. pertenue (T. pertenue) a gene bank of T. pertenue was constructed in lambda vector EMBL3. Clones carrying the T. pertenue gene encoding a 190 kDa protein, TyF1, were selected and the DNA was expressed in E. coli. TyF1 was shown to be closely related, but slightly different from the previously cloned T. pallidum antigen TpF1. TyF1 and TpF1 are high molecular weight antigens of about 190 kDa, which dissociate into 19 kDa subunits after heat treatment in presence of SDS. The difference between the two proteins is most obvious after treatment with proteinase K, which yields a 115 kDa component from TyF1 and a 95 kDa component from TpF1, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The structural genes encoding TyF1 and TpF1 were sequenced and the predicted amino acid sequences differed in a single amino acid residue at position 40, which is arginine in TyF1 and glutamine in TpF1. Similarities TyF1 and TpF1 with the previously described 4D antigen are discussed. The antibody response to TyF1 and TpF1 seems higher in syphilis patients than in yaws patients. The possibility of using the difference between these T. pallidum and the T. Pertenue antigens for serological discrimination of syphilis and yaws is discussed.

Human immunodeficiency virus and tuberculosis coinfection in children: challenges in diagnosis and treatment.

The burden of childhood tuberculosis (TB) is influenced by the human immunodeficiency virus (HIV) epidemic and this dangerous synergy affects various aspects of both diseases; from pathogenesis and the epidemiologic profile to clinical presentation, diagnosis, treatment, and prevention. HIV-infected infants and children are at increased risk of developing severe forms of TB. The TB diagnosis is complicated by diminished sensitivity and specificity of clinical features and diagnostic tools like the tuberculin skin test and chest x-ray. Although alternative ways of pulmonary sampling and the development of interferon-&ggr; assays have shown to lead to some improvement of TB diagnosis in HIV-infected children, new diagnostic tools are urgently needed. Coadministration of anti-TB treatment and antiretroviral drugs induces severe complications, and this highlights the need to define optimal treatment regimens. Practical implementation of these regimens in TB control programs should be combined with isoniazid preventive therapy in TB-exposed HIV-infected children. The risk of severe complications after Bacille Calmette-Guérin vaccination of HIV-infected children emphasizes the need for new nonviable vaccines. This article reviews the current status of pediatric HIV-TB coinfection with specific emphasis on the diagnosis and treatment.

High prevalence of acute respiratory tract infections among Warao Amerindian children in Venezuela in relation to low immunization coverage and chronic malnutrition.

Background: Higher prevalence rates of acute respiratory tract infections (ARTIs) have been described in Australian and Canadian indigenous populations than in nonindigenous age-matched counterparts. Few studies on ARTIs in South American indigenous populations have been published. We performed a cross-sectional survey to describe the prevalence of upper respiratory tract infections and acute lower respiratory tract infections (ALRTIs) and associations with malnutrition and immunization status. Methods: From December 1, 2009 to May 31, 2010, 487 Warao Amerindian children 0 to 59 months of age living in the Delta Amacuro in Venezuela were included in a cross-sectional survey. Data were obtained through parent questionnaires, vaccination cards, and physical examinations including anthropometric measurements. Results: Of the 487 children, 47% presented with an ARTI. Of these, 60% had upper respiratory tract infections and 40% were ALRTI. Immunization coverage was low, with only 27% of all children presenting a vaccination card being fully immunized. The prevalence of malnutrition was high (52%), with stunting (height-for-age <−2 standard deviations) being the most frequent presentation affecting 45% of children. ARTI and ALRTI prevalence diminished with increasing age (odds ratio for ALRTI in children 25–59 months of age vs. children younger than 12 months, 0.49; 95% confidence interval, 0.26–0.93). Furthermore, significant differences in ARTI prevalence were seen between villages. No significant associations between immunization status or malnutrition and ARTI or ALRTI prevalence were identified. Conclusions: A high prevalence of ARTIs and chronic malnutrition in combination with a low immunization status highlights the need for an integrated approach to improve the health status of indigenous Venezuelan children.

A novel quantitative PCR assay for the detection of Streptococcus pneumoniae using the competence regulator gene target comX.

Streptococcus pneumoniae is responsible for an estimated 1.6 million deaths worldwide every year. While rapid detection and timely treatment with appropriate antibiotics is preferred, this is often difficult due to the amount of time that detection with blood cultures takes. In this study, a novel quantitative PCR assay for the detection of Streptococcus pneumoniae was developed. To identify novel targets, we analysed the pneumococcal genome for unique, repetitive DNA sequences. This approach identified comX, which is conserved and present in duplicate copies in Streptococcus pneumoniae but not in other bacterial species. Comparison with lytA, the current gold standard for detection by quantitative PCR, demonstrated an analytic specificity of 100% for both assays on a panel of 10 pneumococcal and 18 non-pneumococcal isolates, but a reduction of 3.5 quantitation cycle values (± 0.23 sem), resulting in an increased analytical detection rate of comX. We validated our assay on DNA extracted from the serum of 30 bacteraemic patients who were blood culture positive for Streptococcus pneumoniae and 51 serum samples that were culture positive for other bacteria. This resulted in a similar clinical sensitivity between the comX and lytA assays (47%) and in a diagnostic specificity of 98.2 and 100% for the lytA and comX assays, respectively. In conclusion, we have developed a novel quantitative PCR assay with increased analytical sensitivity for the detection of Streptococcus pneumoniae, which may be used to develop a rapid bedside test for the direct detection of Streptococcus pneumoniae in clinical specimens.

Childhood Vaccine Acceptance and Refusal among Warao Amerindian Caregivers in Venezuela; A Qualitative Approach.

Objectives Acceptance of childhood vaccination varies between societies, affecting worldwide vaccination coverage. Low coverage rates are common in indigenous populations where parents often choose not to vaccinate their children. We aimed to gain insight into reasons for vaccine acceptance or rejection among Warao Amerindians in Venezuela. Methods Based on records of vaccine acceptance or refusal, in-depth interviews with 20 vaccine-accepting and 11 vaccine-declining caregivers were performed. Parents’ attitudes were explored using a qualitative approach. Results Although Warao caregivers were generally in favor of vaccination, fear of side effects and the idea that young and sick children are too vulnerable to be vaccinated negatively affected vaccine acceptance. The importance assigned to side effects was related to the perception that these resembled symptoms/diseases of another origin and could thus harm the child. Religious beliefs or traditional healers did not influence the decision-making process. Conclusions Parental vaccine acceptance requires educational programs on the preventive nature of vaccines in relation to local beliefs about health and disease. Attention needs to be directed at population-specific concerns, including explanation on the nature of and therapeutic options for side effects.

Nasopharyngeal carriage of respiratory pathogens in Warao Amerindians: significant relationship with stunting.

To assess risk factors for nasopharyngeal carriage of potential pathogens in geographically isolated Warao Amerindians in Venezuela.