TB testing in HIV-positive patients prior to antiretroviral treatment.

BACKGROUND: TB diagnosis in patients with HIV is challenging due to the lower sensitivities across tests. Molecular tests are preferred and the Xpert® MTB/RIF assay has limitations in lower-income settings. We evaluated the performance of loop-mediated isothermal amplification (LAMP) and the lipoarabinomannan (LAM) test in HIV-positive, ART-naïve clinic patients.METHODS: A total of 783 eligible patients were enrolled; three spot sputum samples of 646 patients were tested using TB-LAMP, Xpert, smear microscopy and culture, while 649 patients had TB-LAM testing. Sensitivity, specificity, and negative and positive predictive values were estimated with 95% confidence intervals.RESULTS: Sensitivities for smear microscopy, TB-LAMP and Xpert were respectively 50%, 63% and 74% compared to culture, with specificities of respectively 99.2%, 98.5% and 97.5%. An additional eight were positive on TB-LAM alone. Seventy TB patients (9%) were detected using standard-of-care testing, an additional 27 (3%) were detected using study testing. Treatment was initiated in 57/70 (81%) clinic patients, but only in 56% (57/97) of all those with positive TB tests; 4/8 multidrug-resistant samples were detected using Xpert.CONCLUSION: TB diagnostics continue to miss cases in this high-burden setting. TB-LAMP was more sensitive than smear microscopy, and if followed by culture and drug susceptibility testing as required, can diagnose TB in HIV-positive patients. TB-LAM is a useful add-in test and both tests at the point-of-care would maximise yield.

Detecting Mycobacterium tuberculosis using the loop-mediated isothermal amplification test in South Africa.

SETTING: In South Africa, KwaZulu-Natal is the epicentre of the human immunodeficiency virus (HIV) epidemic, where approximately 70% of people with tuberculosis (TB) are co-infected with HIV. Undiagnosed TB contributes to high mortality in HIV-infected patients. Delays in diagnosing TB and treatment initiation result in prolonged transmission and increased infectiousness. OBJECTIVE: To evaluate the LoopampTM MTBC Detection kit (TB-LAMP; based on the loop-mediated isothermal amplification assay), smear microscopy and Xpert test with the gold standard of mycobacterial culture. METHODS: Sputum samples were collected from 705 patients with symptoms of pulmonary TB attending a primary health care clinic. RESULTS: The TB-LAMP assay had significantly higher sensitivity than smear microscopy (72.6% vs. 45.4%, P < 0.001), whereas specificity was slightly lower (99% vs. 96.8%, P = 0.05), but significantly higher than Xpert (92.9%, P = 0.004). There was no significant difference in sensitivity of smear-positive, culture-positive and smear-negative, culture-positive sputum samples using TB-LAMP vs. Xpert (respectively 95.9%/55.9% vs. 97.6%/66.1%; P =0.65, P = 0.27). The positive predictive value of TB-LAMP was significantly higher than that of Xpert (87.5% vs. 77.0%; P = 0.02), but similar to that of smear microscopy (94.2%; P = 0.18). The negative predictive value was respectively 91.9%, 92.5% (P = 0.73) and 83.1% (P = 0.0001). CONCLUSION: Given its ease of operability, the TB-LAMP assay could be implemented as a point-of-care test in primary health care settings, and contribute to reducing treatment waiting times and TB prevalence.

Targeting of a CD8 T cell env epitope presented by HLA-B*5802 is associated with markers of HIV disease progression and lack of selection pressure.

In HIV-infected persons, certain HLA class I alleles are associated with effective control of viremia, while others are associated with rapid disease progression. Among the most divergent clinical outcomes are the relatively good prognosis in HLA-B*5801 expressing persons and poor prognosis with HLA-B*5802. These two alleles differ by only three amino acids in regions involved in HLA-peptide recognition. This study evaluated a cohort of over 1000 persons with chronic HIV clade C virus infection to determine whether clinical outcome differences associated with B*5801 (n = 93) and B*5802 ( n = 259) expression are associated with differences in HIV-1-specific CD8 (+) T cell responses. The overall breadth and magnitude of HIV-1-specific CD8(+) T cell responses were lower in persons expressing B*5802, and epitope presentation by B*5802 contributed significantly less to the overall response as compared to B*5801-restricted CD8 (+) T cells. Moreover, viral load in B*5802-positive persons was higher and CD4 cell counts lower when this allele contributed to the overall CD8 (+) T cell response, which was detected exclusively through a single epitope in Env. In addition, persons heterozygous for B*5802 compared to persons homozygous for other HLA-B alleles had significantly higher viral loads. Viral sequencing revealed strong selection pressure mediated through B*5801-restricted responses but not through B*5802. These data indicate that minor differences in HLA sequence can have a major impact on epitope recognition, and that selective targeting of Env through HLA-B*5802 is at least ineffectual if not actively adverse in the containment of viremia. These results provide experimental evidence that not all epitope-specific responses contribute to immune containment, a better understanding of which is essential to shed light on mechanisms involved in HIV disease progression.

Effective T-cell responses select human immunodeficiency virus mutants and slow disease progression.

The possession of some HLA class I molecules is associated with delayed progression to AIDS. The mechanism behind this beneficial effect is unclear. We tested the idea that cytotoxic T-cell responses restricted by advantageous HLA class I molecules impose stronger selection pressures than those restricted by other HLA class I alleles. As a measure of the selection pressure imposed by HLA class I alleles, we determined the extent of HLA class I-associated epitope variation in a cohort of European human immunodeficiency virus (HIV)-positive individuals (n=84). We validated our findings in a second, distinct cohort of African patients (n=516). We found that key HIV epitopes restricted by advantageous HLA molecules (B27, B57, and B51 in European patients and B5703, B5801, and B8101 in African patients) were more frequently mutated in individuals bearing the restricting HLA than in those who lacked the restricting HLA class I molecule. HLA alleles associated with clinical benefit restricted certain epitopes for which the consensus peptides were frequently recognized by the immune response despite the circulating virus's being highly polymorphic. We found a significant inverse correlation between the HLA-associated hazard of disease progression and the mean HLA-associated prevalence of mutations within epitopes (P=0.028; R2=0.34). We conclude that beneficial HLA class I alleles impose strong selection at key epitopes. This is revealed by the frequent association between effective T-cell responses and circulating viral escape mutants and the rarity of these variants in patients who lack these favorable HLA class I molecules, suggesting a significant pressure to revert.

Laboratory markers associated with progression of HIV infection.

Infection with HIV may develop to AIDS at different rates in different individuals, with a spectrum varying from rapid progression to long-term non-progression. The variable course of HIV-1 infection causes emotional trauma for the infected person and complicates the design and interpretation of therapeutic trials because of unrecognized differences in prognosis. Owing to the variable clinical expression of HIV infection, the use of non-clinical disease markers has become important to patient management. Thus, it is essential to have tests which can accurately assess the stage of infection in an individual, as well as predict its course and monitor its progression. These laboratory tests are valuable during the period of clinical latency and subsequently supplement various clinical parameters.

HIV evolution: CTL escape mutation and reversion after transmission.

Within-patient HIV evolution reflects the strong selection pressure driving viral escape from cytotoxic T-lymphocyte (CTL) recognition. Whether this intrapatient accumulation of escape mutations translates into HIV evolution at the population level has not been evaluated. We studied over 300 patients drawn from the B- and C-clade epidemics, focusing on human leukocyte antigen (HLA) alleles HLA-B57 and HLA-B5801, which are associated with long-term HIV control and are therefore likely to exert strong selection pressure on the virus. The CTL response dominating acute infection in HLA-B57/5801-positive subjects drove positive selection of an escape mutation that reverted to wild-type after transmission to HLA-B57/5801-negative individuals. A second escape mutation within the epitope, by contrast, was maintained after transmission. These data show that the process of accumulation of escape mutations within HIV is not inevitable. Complex epitope- and residue-specific selection forces, including CTL-mediated positive selection pressure and virus-mediated purifying selection, operate in tandem to shape HIV evolution at the population level.

Characterisation of the pncA gene in Mycobacterium tuberculosis isolates from Gauteng, South Africa.

SETTING: The use of pyrazinamide (PZA) is important for the treatment of Mycobacterium tuberculosis as it is bactericidal to semi-dormant mycobacteria that are not affected by other drugs. The incidence of resistance to PZA and other drugs used in the treatment of M. tuberculosis is increasing in South Africa. OBJECTIVE: To characterise the pncA gene of M. tuberculosis isolates from Gauteng, South Africa, and to develop a rapid diagnostic method. DESIGN: The pncA gene and the putative regulatory gene were characterised by sequence analysis in a total of six PZA susceptible and 15 resistant isolates. The association with classical PZA susceptibility testing and PZase activity was determined. RESULTS: All PZA-resistant isolates were PZase negative as well as resistant to at least one other anti-tuberculosis drugs. Mutations were identified throughout the length of the pncA gene in 10/15 PZA-resistant isolates. Five lacked PZase activity, but the wild type pncA sequence was present. In all six PZase-positive strains, a PZA-susceptible pattern was obtained on BACTEC and the wild type pncA sequence was present. CONCLUSION: Sequencing is an effective means to identify mutations in the pncA gene in M. tuberculosis and therefore resistance to PZA. The fact that some PZA-resistant M. tuberculosis isolates lack mutations in the pncA gene suggests that alternative mechanisms for drug resistance exist. In PZase negative strains with no genetic changes which are resistant to 100 microg/ml and susceptible to 300 microg/ml, 300 microg/ml may be a more reliable breakpoint.

Genomic mutations in the katG, inhA and aphC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa.

Genotypic analysis of isoniazid (INH) resistance in 79 isolates of M. tuberculosis (MTB) was undertaken by PCR-single strand conformation polymorphism (SSCP), Msp1 restriction enzyme analysis and sequence analysis of specific regions of three genes (part of the coding sequence of katG, and promoter regions of the inhA operon and ahpC) in order to determine the particular allelic variants within these genes. The epidemiologic relatedness was determined using IS6110 and polymorphic G-C region (PGRS (MTB484(1)) based restriction fragment length polymorphism (RFLP). Mutations in katG, inhA locus and ahpC were identified in 77/79, 19/79 and 10/79 isolates respectively. The ability of PCR-SSCP to detect mutations associated with INH resistance in katG, inhA and ahpC genes was 100% (CI 91.2-99.7%), 98.7% (CI 74.0-99.9%), and 100% (CI 69.2-100%) respectively. Specificity was 100%. All isolates with mutations in the 209 bp fragment of the MTB katG gene containing the Ser315Thr codon were positive by PCR-RFLP using Msp1 enzyme restriction analysis. Sixteen of 19 isolates with alterations on the 3' end of the ribosome binding site upstream of mabA in inhA locus simultaneously harbored Ser315Thr mutations in KatG. In 9/10 isolates, mutations in the ahpC promoter region were located in the 105 bp oxyR-ahpC intergenic region. None of 17 INH drug susceptible isolates harbored mutations in any of the three genetic regions, although the katG1 allele (Arg 463 Leu) was present in one isolate. Characterization by IS6110/PGRS(MTB484(1))RFLP analysis revealed that a number of drug resistant clones are widespread in the community. We conclude that the frequency of the Ser315Thr katG mutation in the local strain population makes the PCR-RFLP MTB katG assay a reliable, rapid and useful method for detecting INH resistance.

Rapid screening of Mycobacterium tuberculosis for susceptibility to rifampicin and streptomycin.

OBJECTIVE: To investigate rapid detection of drug-resistant tuberculosis using the genotypic Inno-LiPA Rif TB assay and a novel, low-cost, bacteriophage-based susceptibility assay. DESIGN: The performance of the microwell phage replication assay (MPRA) on 18 isolates from suspected multidrug-resistant tuberculosis patients was compared to the LiPA assay performed directly on sputum specimens. Mutations in the rpoB gene identified by LiPA that confer resistance to rifampicin (RMP) were confirmed by DNA sequencing, while susceptibilities were confirmed by the proportion method and BACTEC. A further 19 isolates undergoing routine screening for both RMP and streptomycin susceptibility were included for comparison. RESULTS: Susceptibility to RMP was determined for 17/18 (94.4%) sputum specimens tested by LiPA. Correlation between MPRA, molecular and conventional methods was 100% for the detection of RMP susceptibility. However, for susceptibility to streptomycin one discrepant result was found: an isolate susceptible to streptomycin by the proportion method was found resistant by MPRA to 2 microg/ml of streptomycin. Similarly, an isolate initially resistant by MPRA upon re-testing was found susceptible in agreement with the conventional method. CONCLUSION: LiPA enables rapid detection of drug-resistant infection, while MPRA offers simple, low-tech testing of drug susceptibilities that may be appropriate for application in low-income countries.

Phylogenetic analysis of Calymmatobacterium granulomatis based on 16S rRNA gene sequences.

Calymmatobacterium granulomatis is the aetiological agent of granuloma inguinale - a chronic granulomatous genital infection - and is morphologically similar to members of the genus Klebsiella. This study determined the 16S rRNA gene sequence of C. granulomatis and the taxonomic position of the organism in relation to the genus Klebsiella. Genomic DNA was extracted from C. granulomatis-infected monocytes and from frozen and formalin-fixed paraffin wax-embedded tissue biopsy specimens from patients with histologically proven granuloma inguinale. The 16S rDNA was amplified by PCR with broad range oligonucleotide primers. The amplified DNA fragments were cloned into pMOS vector, digested with Bam HI and Pst1 restriction endonucleases, hybridised with a gram-negative bacterial probe (DL04), sequenced in both directions by the automated ALF DNA sequencer, verified on an ABI Prism 377 automated sequencer and analysed with DNASIS and MEGA software packages. Sequence analysis revealed DNA homology of 99% in C. granulomatis from the different sources, supporting the belief that the bacteria in the culture and the biopsy specimens belonged to the same species, although there was some diversity within the species. Phylogenetically, the strains were closely related to the genera Klebsiella and Enterobacter with similarities of 95% and 94% respectively. C. granulomatis is a unique species, distinct from other related organisms belonging to the gamma subclass of Proteobacteria.

Ultrastructure of Calymmatobacterium granulomatis: comparison of culture with tissue biopsy specimens.

The ultrastructural features of cells of Calymmatobacterium granulomatis from monocyte co-cultures and tissue biopsy specimens were compared. In cultures the bacteria were mainly extracellular, i.e., not within membrane-bound vacuoles. The bacterial body was surrounded by a uniformly extensive homogeneous layer with a relatively high electron density. This layer varied considerably in tissue biopsy specimens, having either homogeneously electron-dense or delicate web-like structures with varying density and thickness. In tissue specimens the bacteria were located predominantly within vacuoles of varying sizes in the cytoplasm of the macrophages and, occasionally, extracellularly within the intercellular spaces of the stroma. The bacterial cytoplasm contained ribosomes scattered throughout with electron-dense granules located peripherally. The trilaminar cell-wall structure was typical of a gram-negative organism, comprising an outer membrane, a middle electron-opaque layer and an inner plasma membrane. Surface structures such as fimbriae, flagella and bacteriophages were not identified in specimens from either source.

Comparison of PCR-heteroduplex characterization by automated DNA sequencing and line probe assay for the detection of rifampicin resistance in Mycobacterium tuberculosis isolates from KwaZulu-Natal, South Africa.

Progress in understanding the basis of resistance to rifampicin (RifR) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. One hundred thirteen strains of Mycobacterium tuberculosis isolated from patients with multidrug resistant tuberculosis (MDR-TB) were investigated for genotypic analysis of RifR by polymerase chain reaction-heteroduplex formation (PCR-HDF) and characterization of mutations by automated DNA sequencing of the rpoB gene. A subset of isolates (22) representative of different mutations as confirmed by sequence analysis were also evaluated by the Line Probe Assay (LiPA). In 106 of the RifR strains, 24 mutations within an 81-bp region of the rpoB gene affecting 13 amino acids were observed. Most isolates (7/8) harboring Leu533 --> Pro codon mutation required minimum inhibitory concentrations (MICs) of < or = 8 microg/ml. There was geographic variation in the frequency of occurrence of particular rpoB mutations, with the Ser531 --> Leu/Trp codon mutation found in 59/113 of isolates. Although there are certain limitations in the use of both the rapid PCR-HDF diagnostic assay and the LiPA for the detection of rifampicin susceptibility of M. tuberculosis, these provide important and convenient tools for identifying and managing patients with MDR-TB.

Growth and cultural characteristics of Calymmatobacterium granulomatis--the aetiological agent of granuloma inguinale (Donovanosis).

Granuloma inguinale is a chronic destructive granulomatous disease of the genitalia. The clinical diagnosis is often unreliable and the definitive diagnosis is based on the visualisation of 'Donovan bodies' in tissue smears or biopsy specimens. The organism implicated in its aetiology, Calymmatobacterium granulomatis, was reported to have been cultured > 30 years ago, but little is known about the organism because of its fastidious nature and the difficulty in culturing it. Twenty-two biopsy specimens from female patients with clinical and laboratory-confirmed granuloma inguinale were treated with amikacin 10 mg/L and inoculated in a monocyte co-culture system with peripheral blood mononuclear cells (PBMC) from a single donor and autologous sera. The method was subsequently modified by pretreatment of specimens with vancomycin 5 mg/L and metronidazole 10 mg/L in addition to amikacin 10 mg/L for the purpose of decontamination, pooled blood donor PBMC and by the use of heat-inactivated fetal calf serum instead of autologous serum for culture. This modified method was used to culture additional biopsy specimens and genital ulcer scrapings from female and male patients, respectively. All monocyte co-cultures were examined by a rapid Giemsa (RapiDiff) stain and by an indirect immunofluorescence test with immune sera. Representative cultures were examined by transmission electron microscopy. C. granulomatis was successfully isolated in pure culture by the monocyte co-culture system from four biopsy specimens and 14 genital ulcer scrapings. The cultured organisms were visible both intra- and extra-cellularly and were extremely pleomorphic, with characteristic single and biopolar condensation. The numbers of the organisms increased after each passage. All positive cultures showed bright fluorescence when tested with immune sera. Transmission electron microscopy of the cultured bacteria demonstrated a typical gram-negative cell wall consisting of an outer membrane, middle electron opaque layer and an inner plasma membrane. The capsule was thick and electron dense. Numerous electron dense granules were present within the cytoplasm.

Ribosomal DNA typing of Haemophilus ducreyi strains: proposal for a novel typing scheme.

Local strains of Haemophilus ducreyi were characterized by ribosomal DNA fingerprinting in order to develop a typing scheme for this organism. Chromosomal DNAs from 30 strains were extracted, and restriction fragments were separated by agarose gel electrophoresis after cleavage with BglII, HindIII, HindII, and BstEII. Of the 30 local strains studied, nine BglII, eight HindIII, six HindII, and five BstEII ribotype patterns were observed. In order to develop a typing scheme, we correlated the ribotype patterns obtained with each of the four enzymes for each strain. By this scheme, 13 ribotypes designated b to n were defined among the local strains. This study demonstrates that there is a diversity of ribotypes among H. ducreyi strains in our area, which emphasizes that there are many strains responsible for chancroid. This novel typing scheme provides a useful tool for further epidemiological studies of chancroid.

Type II human complement C2 deficiency. Allele-specific amino acid substitutions (Ser189 --> Phe; Gly444 --> Arg) cause impaired C2 secretion.

Type II complement protein C2 deficiency is characterized by a selective block in C2 secretion. The Type II C2 null allele (C2Q0) is linked to two major histocompatibility haplotypes (MHC) that differ from the MHC of the more common Type I C2 deficiency. To determine the molecular basis of Type II deficiency the two Type II C2Q0 genes were isolated and transfected separately into L-cells. Subsequent molecular biology, biosynthetic, and immunofluorescence studies demonstrated that C2 secretion is impaired in Type II C2 deficiency because of different missense mutations at highly conserved residues in each of the C2Q0 alleles. One is in exon 5 (nucleotide C566 --> T; Ser189 --> Phe) of the C2Q0 gene linked to the MHC haplotype A11,B35,DRw1,BFS, C4A0B1. The other is in exon 11 (G1930 --> A; Gly444 --> Arg) of the C2Q0 gene linked to the MHC haplotype A2,B5, DRw4,BFS,C4A3B1. Each mutant C2 gene product is retained early in the secretory pathway. These mutants provide models for elucidating the C2 secretory pathway.

Vitamin A supplementation enhances specific IgG antibody levels and total lymphocyte numbers while improving morbidity in measles.

The effect of vitamin A supplementation on selected factors of immunity was tested in African children (ages 4 to 24 months with complicated measles) during a randomized double-blind intervention trial. Placebo (n = 31) and treated groups (n = 29) had similar baseline characteristics. The supplemented group had significant reductions in morbidity (expressed as integrated morbidity scores) during the acute (Day 8, P = 0.006) and chronic (Day 42, P = 0.02; 6 months; P = 0.002) phases. In the treated group there was an increase in total number of lymphocytes (Day 42, P = 0.05) and measles IgG antibody concentrations (Day 8, P = 0.02), both of which have consistently been previously shown to correlate more closely with outcome in measles than other immunologic, clinical and radiologic factors. Interleukin 2 and plasma complement values were unaffected by vitamin A supplementation. These findings reinforce results from animal studies that show that the pathways of vitamin A activity in decreasing morbidity and mortality are partly founded on selective immunopotentiation.

Loss of maternal measles antibody in black South African infants in the first year of life--implications for age of vaccination.

In order to investigate the feasibility of measles vaccination before the age of 9 months the duration of passive immunity against measles was estimated by conducting a longitudinal study of measles antibody levels in 20 black neonates delivered at term. Measles serum antibody (IgG) was measured by enzyme-linked immunosorbent assay in the mother at childbirth and on consecutive samples taken from the infants from birth until 9 months of age. Protective measles antibody level was defined as greater than 200 mIU. Unprotective levels were found in 88% (95% confidence interval (CI) 81-99%) of 6-month-old infants, while at 9 months all were susceptible. The mean antibody level was 192 mIU (CI 104-348%) at 4 months; 34 mIU (CI 15-73%) at 6 months and 13 mIU (CI 6-24%) at 9 months of age. Our data support the recent World Health Organisation recommendation to immunise children in developing countries at 6 months with the 'high titre' Edmonston-Zagreb measles vaccine, since most infants in our study had lost passive immunity against measles by this age.

Lack of efficacy of the standard potency Edmonston-Zagreb live, attenuated measles vaccine in African infants.

The efficacy of standard potency Edmonston-Zagreb (E-Z) measles vaccine was tested in a randomized trial of Black infants in a rural area of South Africa where a measles epidemic was occurring. The following immunization schedules were used: 48 infants aged 4-8.5 months who received 3.9 log 50 infectious units of E-Z vaccine (group A); 48 infants aged 4-8.5 months who received 3.28 log 50 infectious units of Schwarz vaccine (group B); and 28 infants aged greater than 9 months who received 3.28 log 50 infectious units of Schwarz vaccine and served as controls (group C). For infants aged less than 23 weeks who were given either the E-Z or Schwarz vaccine, the number of seropositives was low (28%), irrespective of the pre-vaccination level of measles antibody. There was a higher number of seropositives (68%) among those in the age range greater than 23 weeks to less than 36 weeks who received the E-Z vaccine rather than the Schwarz vaccine (36%). When administered to children aged greater than 36 weeks, the Schwarz vaccine produced a satisfactory, though suboptimal response rate (61%). There was no correlation between seropositivity and pre-vaccination measles antibody status. Use of the standard dose of E-Z vaccine may have been one of the factors for this poor response, and this supports the WHO recommendation that titres higher than the standard potency vaccine are needed if 6-month-old infants are to be successfully immunized against measles.