Increase in anti-tuberculosis drug resistance in Botswana: results from the fourth National Drug Resistance Survey.

SETTING: Although approximately 0.5 million cases of multidrug-resistant tuberculosis (MDR-TB) occur globally each year, surveillance data are limited. Botswana is one of the few high TB burden countries to have carried out multiple anti-tuberculosis drug resistance surveys (in 1995-1996, 1999 and 2002). OBJECTIVE: In 2007-2008, we conducted the fourth national survey of anti-tuberculosis drug resistance in Botswana to assess anti-tuberculosis drug resistance, including trends over time. In the previous survey, 0.8% (95%CI 0.4-1.5) of new patients and 10.4% (95%CI 5.6-17.3) of previously treated patients had MDR-TB. DESIGN: During the survey period, eligible specimens from all new sputum-smear positive TB patients and from all TB patients with history of previous anti-tuberculosis treatment underwent mycobacterial culture and anti-tuberculosis drug susceptibility testing (DST). RESULTS: Of 924 new TB patients and 137 with previous anti-tuberculosis treatment with DST results, respectively 23 (2.5%, 95%CI 1.6-3.7) and 9 (6.6%, 95%CI 3.3-11.7) had MDR-TB. The proportion of new TB patients with MDR-TB has tripled in Botswana since the previous survey. CONCLUSION: Combatting drug-resistant TB will require the scale-up of MDR-TB diagnosis and treatment to prevent the transmission of MDR-TB and strengthening of general TB control to prevent the emergence of resistance.

Anti-tuberculosis treatment outcomes in HIV-infected adults exposed to isoniazid preventive therapy in Botswana.

SETTING: Eight public health clinics in Gaborone and Francistown, Botswana. OBJECTIVES: To describe the characteristics and outcomes of incident tuberculosis (TB) cases in human immunodeficiency virus (HIV) infected adults exposed to isoniazid preventive therapy (IPT) with access to antiretroviral and anti-tuberculosis treatment. DESIGN: In 1995 HIV-infected adults, TB disease was excluded before commencing IPT. During and after receipt of 6 or 36 months of IPT, symptomatic participants were evaluated using chest radiographs, sputum microscopy, cultures and drug susceptibility testing (DST). Incident TB cases received ≥6 months of anti-tuberculosis treatment. RESULTS: Seventy-five incident TB cases were identified among 619 symptomatic participants. The median duration of IPT in these cases was 6 months (range 1-35), and the median time to initiation of anti-tuberculosis treatment was 12 months after IPT cessation. Antiretroviral therapy (ART) was initiated before anti-tuberculosis treatment in 37 cases. Culture was positive in 43/58 (74%) TB cultures. DST was available for 38 cases, of which six (16%) were resistant to isoniazid (INH); 67/75 (89%) cases, including four with INH-monoresistant TB, completed anti-tuberculosis treatment or were cured. CONCLUSIONS: With prompt initiation of anti-tuberculosis treatment and access to ART, excellent outcomes were achieved in a public health setting in HIV-infected adults who developed TB disease.

Association of chest radiographic abnormalities with tuberculosis disease in asymptomatic HIV-infected adults.

SETTING: Francistown and Gaborone, Botswana. OBJECTIVE: Chest radiography is used to screen for tuberculosis (TB) in asymptomatic persons living with the human immunodeficiency virus (PLWH) seeking isoniazid preventive therapy (IPT). We describe radiographic features in PLWH in a TB-endemic setting and identify features associated with TB disease. DESIGN: Asymptomatic PLWH seeking IPT under program conditions for a clinical trial between 2004 and 2006 received chest radiographs (CXRs) that were read using the standardized Chest Radiograph Reading and Recording System (CRRS). Clinical characteristics, including TB disease, were compared with the radiographic findings. RESULTS: From 2732 screening CXRs, 183 had one or more abnormalities and were scored using CRRS, with 42% having infiltrates (36% upper lobes), 35% parenchymal fibrosis and 32% adenopathy. TB disease status was determined in 129 (70%) PLWH, of whom 22 (17%) had TB disease. TB disease was associated with upper lobe infiltrates (relative risk [RR] 3.0, 95%CI 1.5-6.2) and mediastinal adenopathy (RR 3.9, 95%CI 1.8-8.4). The sensitivity and specificity of either upper lobe infiltrates or mediastinal lymphadenopathy for TB disease were respectively 64% and 82%. CONCLUSION: A combination of CXR features was useful for predicting TB disease in asymptomatic PLWH. CRRS should be used more frequently in similar studies.

Tuberculosis in asymptomatic HIV-infected adults with abnormal chest radiographs screened for tuberculosis prevention.

BACKGROUND: Isoniazid preventive therapy (IPT) prevents tuberculosis (TB) in people living with HIV (human immunodeficiency virus, PLWH). Symptom screening without chest radiographs (CXRs) was established as the strategy for excluding TB disease among PLWH seeking IPT in Botswana's 2001 pilot project. This strategy was evaluated in 2004-2006 among candidates screened for an IPT clinical trial. METHODS: PLWH referred from clinics and HIV testing centers were screened for TB symptoms. All asymptomatic candidates received CXRs; those with abnormal CXRs were investigated further. RESULTS: Among 2732 asymptomatic candidates screened, 302 (11%) had abnormal CXRs potentially compatible with TB; TB disease was diagnosed in 43 of these 302 (14%), or 43 (1.6%) of the 2732 asymptomatic candidates. While not associated with CD4 lymphocyte counts < 200 cells/mm(3), TB was associated with a positive tuberculin skin test (relative risk 2.1, 95%CI 1.1-4.0). IPT was initiated in 113 (62%) of 182 asymptomatic PLWH with abnormal CXRs; 8/113 (7%) subsequently developed TB, and 7/8 (88%) successfully completed anti-tuberculosis treatment. CONCLUSIONS: The prevalences of abnormal CXRs and TB were respectively 2.6- and 8.9-fold higher among asymptomatic PLWH screened for the trial than in the pilot. A cost-effectiveness analysis is needed to determine whether the benefits of symptom screening alone are offset by the risk of inducing INH resistance by excluding CXRs during screening.

Baseline evaluation of routine HIV testing among tuberculosis patients in Botswana.

In January 2004, the government of Botswana introduced a policy of routine, non-compulsory human immunodeficiency virus (HIV) testing to increase testing and access to antiretroviral treatment (ART) for individuals presenting for medical treatment. Before a systematic implementation of the policy, we conducted a cross-sectional survey of tuberculosis (TB) record data from 46 clinics in 10 districts to assess baseline HIV testing rates among TB patients. Recorded HIV results from the facility TB register and TB treatment card were reviewed. Of the 1242 TB patients entered in the register, 47% had a recorded HIV result and 84% of these were co-infected with HIV. TB treatment cards were available for 862 (69%) registered patients. Among the 411 (47%) with test results recorded on the treatment card, 341 (83%) were HIV-infected; of these, 12% were reported to be receiving ART.

Reported childhood tuberculosis treatment outcomes, Gaborone and Francistown, Botswana, 1998-2002.

SETTING: Botswana. OBJECTIVES: To estimate frequencies of tuberculosis (TB) treatment outcomes, assess the validity of reported treatment outcomes, and identify risk factors for death during TB treatment among children aged <15 years during 1998-2002. DESIGN: We examined TB treatment outcome frequencies using the national Electronic TB Registry (ETR) data. Treatment and medical records were reviewed to calculate predictive values (PV) for outcomes recorded in the ETR. We interviewed parents of children treated for TB and assessed risk factors for death during treatment via case-control study. RESULTS: Of 5483 patients, 3646 (67%) were cured or completed treatment and 577 (10.5%) died during treatment. The PV for ETR was 76% for death and 97% for cured or completed treatment. We interviewed parents of 91 children who died during treatment and 220 children who completed treatment. Human immunodeficiency virus (HIV) status was unknown for 76% of the children and 54% of the parents. Parent-reported adverse effects to anti-tuberculosis medication (adjusted odds ratio [aOR] 4.9, 95% confidence limit [CL] 2.2-9.2), and lower patient age (aOR 2.2, 95%CL 1.2-4.2) were associated with death during treatment. CONCLUSIONS: TB control programs in Botswana should assess for potential adverse effects of anti-tuberculosis medication and expand HIV testing among children with TB and their parents.

Shigella flexneri 2a strain CVD 1207, with specific deletions in virG, sen, set, and guaBA, is highly attenuated in humans.

A phase 1 clinical trial was conducted among 35 healthy adult volunteers to evaluate the safety, immunogenicity, and shedding of different doses of CVD 1207, a live attenuated Shigella flexneri 2a vaccine candidate with specific deletion mutations in virG, sen, set, and guaBA. CVD 1207 retains the ability to invade epithelial cells but cannot effectively spread intercellularly after invasion (DeltavirG), does not produce enterotoxin (Deltasen and Deltaset), and has limited proliferation in vivo (DeltaguaBA). In a consecutive fashion, groups of three to seven subjects ingested a single oral dose of CVD 1207 at an inoculum of either 10(6), 10(7), 10(8), 10(9), or 10(10) CFU. CVD 1207 was remarkably well-tolerated at inocula as high as 10(8) CFU. In comparison, one of 12 subjects who received 10(9) CFU experienced mild diarrhea and another experienced a single episode of emesis. One of five subjects who received 10(10) CFU experienced watery diarrhea and emesis. All subjects who ingested doses of 10(8) to 10(10) CFU excreted the vaccine; in 23 of 25, the duration of excretion was

Production of IFN-gamma and IL-10 to Shigella invasins by mononuclear cells from volunteers orally inoculated with a Shiga toxin-deleted Shigella dysenteriae type 1 strain.

Volunteers were orally administered invasive, non-Shiga toxin-producing Shigella dysenteriae 1 to establish a challenge model to assess vaccine efficacy. In stepwise fashion, four separate groups were given 3 x 10(2), 7 x 10(3), 5 x 10(4), or 7 x 10(5) CFU. Using PBMC, proliferative responses and cytokine production were measured to S. dysenteriae whole-cell preparations and to purified recombinant invasion plasmid Ags (Ipa) C and IpaD. Anti-LPS and anti-Ipa Abs and Ab-secreting cells were also evaluated. Preinoculation PBMC produced considerable quantities of IL-10 and IFN-gamma, probably secreted by monocytes and NK cells, respectively, of the innate immune system. Following inoculation, PBMC from 95 and 87% of volunteers exhibited an increased production of IFN-gamma and IL-10, respectively, in response to Shigella Ags. These increases included responses to IpaC and IpaD among those volunteers receiving the lowest inoculum. No IL-4 or IL-5 responses were detected. Whereas there were no Ab or Ab-secreting cell responses in volunteers receiving the lowest inoculum, other dose groups had moderate to strong anti-LPS and anti-Ipa responses. These results suggest that in humans, type 1 responses play an important role in mucosal and systemic immunity to S. dysentariae 1.

A study of the effects of altering the sites for N-glycosylation in alpha-1-proteinase inhibitor variants M and S.

alpha-1-Proteinase inhibitor (A1Pi) is a monomeric secreted protein glycosylated at asparagines 46, 83, and 247. For this study cDNAs for M (normal) and S (Glu264-->Val) variants of A1Pi were altered by site-directed mutagenesis to produce the combinations of single, double, and triple mutants that can be generated by changing the codons normally specifying these Asn residues to encode Gln. The fates of the mutant proteins were followed in transiently transfected COS-1 cells. All variants with altered glycosylation sites are secreted at reduced rates, are partially degraded, accumulate intracellularly, and some form Nonidet P-40-insoluble aggregates. The carbohydrate attached at Asn83 seems to be of particular importance to the export of both A1PiM and A1PiS from the endoplasmic reticulum. All mutations affecting glycosylation of A1PiS notably reduce secretion, cause formation of insoluble aggregates, and influence degradation of the altered proteins. The variant of A1PiS missing all three glycosylation sites is poorly secreted, is incompletely degraded, and accumulates in unusual perinuclear vesicles. These studies show that N-linked oligosaccharides in A1Pi are vital to its efficient export from the endoplasmic reticulum and that the consequences of changing the normal pattern of glycosylation vary depending upon the sites altered and the variant of A1Pi bearing these alterations.

Effects of mutations that alter the Glu264-Lys387 salt bridge on the secretion of alpha-1-proteinase inhibitor.

The possibility that low circulating levels of alpha-1-proteinase inhibitor (A1Pi) in individuals homozygous for the S variant result from disruption of a salt bridge between Glu264 and Lys387 was examined. Mutations effecting this salt bridge were constructed by either altering the common M variant cDNA so that Glu264 was replaced by Val to produce the S variant (A1PiV264) or Lys387 was replaced by Arg (A1PiR387), Asn (A1PiN387), Glu (A1PiE387), Gly (A1PiG387), Ile (A1PiI387), or Leu (A1PiL387). Measurements of secretion from transiently transfected COS cells showed that A1PiG387 and A1PiI387 are secreted as well as and at least as rapidly as A1PiM; the S variant and A1PiL387 are secreted to about the same extent as, but somewhat more slowly, than A1PiM; and the variants containing polar or charged residues at position 387 are poorly secreted, and unlike the other variants in this series undergo significant degradation by 2 h of chase. We conclude that the low circulating level of A1PiS is not due to inefficient secretion nor to excessive intracellular degradation of this variant. In addition we suspect that the lack of secretion of variants with Lys387 replaced by other charged or polar residues is due to alteration of a highly conserved sequence near the carboxyl terminus of A1Pi.