Circulating microbial products and acute phase proteins as markers of pathogenesis in lymphatic filarial disease.

Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Dysregulated host inflammatory responses leading to systemic immune activation are thought to play a central role in filarial disease pathogenesis. We measured the plasma levels of microbial translocation markers, acute phase proteins, and inflammatory cytokines in individuals with chronic filarial pathology with (CP Ag+) or without (CP Ag-) active infection; with clinically asymptomatic infections (INF); and in those without infection (endemic normal [EN]). Comparisons between the two actively infected groups (CP Ag+ compared to INF) and those without active infection (CP Ag- compared to EN) were used preliminarily to identify markers of pathogenesis. Thereafter, we tested for group effects among all the four groups using linear models on the log transformed responses of the markers. Our data suggest that circulating levels of microbial translocation products (lipopolysaccharide and LPS-binding protein), acute phase proteins (haptoglobin and serum amyloid protein-A), and inflammatory cytokines (IL-1ß, IL-12, and TNF-α) are associated with pathogenesis of disease in lymphatic filarial infection and implicate an important role for circulating microbial products and acute phase proteins.

Toll-like receptor- and filarial antigen-mediated, mitogen-activated protein kinase- and NF-κB-dependent regulation of angiogenic growth factors in filarial lymphatic pathology.

Filarial lymphatic pathology is of multifactorial origin, with inflammation, lymphangiogenesis, and innate immune responses all playing important roles. The role of Toll-like receptors (TLRs) in the development of filarial pathology is well characterized. Similarly, the association of pathology with elevated levels of plasma angiogenic factors has also been documented. To examine the association between TLR function and the development of lymphangiogenesis in filarial infections, we examined TLR- and filarial antigen-induced expression and production of various angiogenic growth factors. We demonstrate that TLR ligands (specifically TLR2, -3, and -5 ligands) induce significantly increased expression/production of vascular endothelial growth factor A (VEGF-A) and angiopoietin-1 (Ang-1) in the peripheral blood mononuclear cells of individuals with lymphatic pathology (CP individuals) compared to that in cells of asymptomatic infected (INF) individuals. Similarly, filarial antigens induce significantly enhanced production of VEGF-C in CP compared with INF individuals. TLR2-mediated enhancement of angiogenic growth factor production in CP individuals was shown to be dependent on mitogen-activated protein kinase (MAPK) and NF-κB signaling, as pharmacologic inhibition of either extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, or NF-κB signaling resulted in significantly diminished production of VEGF-A and Ang-1. Our data therefore strongly suggest an important association between TLR signaling and lymphangiogenesis in the development of pathology in human lymphatic filariasis.

Filarial lymphatic pathology reflects augmented toll-like receptor-mediated, mitogen-activated protein kinase-mediated proinflammatory cytokine production.

Lymphatic filariasis can be associated with the development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Toll-like receptors (TLRs) are thought to play a major role in the development of filarial pathology. To elucidate the role of TLRs in the development of lymphatic pathology, we examined cytokine responses to different Toll ligands in patients with chronic lymphatic pathology (CP), infected patients with subclinical pathology (INF), and uninfected, endemic-normal (EN) individuals. TLR2, -7, and -9 ligands induced significantly elevated production of Th1 and other proinflammatory cytokines in CP patients in comparison to both INF and EN patients. TLR adaptor expression was not significantly different among the groups; however, both TLR2 and TLR9 ligands induced significantly higher levels of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein (MAP) kinases (MAPK) as well as increased activation of NF-κB in CP individuals. Pharmacologic inhibition of both ERK1/2 and p38 MAP kinase pathways resulted in significantly diminished production of proinflammatory cytokines in CP individuals. Our data, therefore, strongly suggest an important role for TLR2- and TLR9-mediated proinflammatory cytokine induction and activation of both the MAPK and NF-κB pathways in the development of pathology in human lymphatic filariasis.

Acquired alpha 1-antitrypsin deficiency in tropical pulmonary eosinophilia.

BACKGROUND & OBJECTIVES: Observation of an increased frequency of an intermediate deficiency of serum alpha1-antitrypsin (α1-AT) in patients with Tropical Pulmonary Eosinophilia (TPE) was earlier reported. Though the possibility of existence of an acquired deficiency was suggested, without phenotyping a hereditary α1-AT deficiency in TPE could not totally be ruled out. In this study, we have done Pi (Protease inhibitor) phenotyping to investigate the possibility of association of any heterozygous (or homozygous) α1-AT deficiency in patients with TPE. METHODS: Serum a1antitrypsin (α1-AT) was measured in 103 patients (Group A) with TPE, 99 patients with pulmonary eosinophilia who had associated intestinal worm infestation (Group B) and 43 healthy volunteers who served as controls. In 19 α1-AT deficient patients (9 of Group A and 10 of Group B), α1-AT level was measured before and after treatment. In 58 patients with TPE and in 5 controls, phenotyping was done. RESULTS: Fifteen patients of Group A and 16 from Group B showed intermediate α1-AT deficiency (150 mg % or less. None of the control subjects had α1-AT deficiency (<200 mg%). After treatment with DEC and/or deworming, in 19 patients there was a significant (P < 0.001) rise in α1-AT levels. Results of phenotyping showed that all had M1 or M 2 allele and none had S or Z variant (either homozygous or heterozygous) thus ruling out any underlying genetic cause for the observed α1-AT deficiency. INTERPRETATION & CONCLUSIONS: The observed α1-AT deficiency may be due to the chronic inflammation in TPE and associated oxidative stress. However, in such α1-AT deficient patients with TPE and those with worm infested pulmonary eosinophilia, faecal α1-AT concentration and faecal α1-AT clearance should be routinely estimated to rule out the possibility of any intestinal protein loss.

Diurnally subperiodic filariasis in India-prospects of elimination: precept to action?

The elimination of lymphatic filariasis in the Andaman and Nicobar Islands provides unique opportunities and challenges at the same time. Since these islands are remote, are sparsely populated, and have poor transport networks, mass drug administration programs are likely to be difficult to implement. Diurnally subperiodic Wuchereria bancrofti vectored by Downsiomyia nivea was considered for the scope of vector control options. Considering the bioecology of this mosquito, vector control including personal protection measures may not be feasible. However, since these islands are covered by separate administrative machinery which also plays an important role in regulating the food supply, the use of diethylcarbamazine (DEC)-fortified salt as a tool for the interruption of transmission is appealing. DEC-fortified salt has been successfully pilot tested in India and elsewhere, operationally used by China for eliminating lymphatic filariasis. Administration of DEC-fortified salt though simple, rapid, safe, and cost-effective, challenges are to be tackled for translating this precept into action by evolving operationally feasible strategy. Although the use of DEC-fortified salt is conceptually simple, it requires commitment of all sections of the society, an elaborate distribution mechanism that ensures the use of DEC-fortified salt only in the endemic communities, and a vigorous monitoring mechanism. Here, we examine the inbuilt administrative mechanisms to serve the tribal people, health infrastructure, and public distribution system and discuss the prospects of putting in place an operationally feasible strategy for its elimination.

Regulatory T cells modulate Th17 responses in patients with positive tuberculin skin test results.

BACKGROUND: The factors governing latency in tuberculosis are not well understood but appear to involve both the pathogen and the host. We have used tuberculin skin test (TST) positivity as a tool to study cytokine responses in latent tuberculosis. METHODS: To identify the host factors that are important in the maintenance of TST positivity, we examined mycobacteria-specific immune responses of TST-positive (latent tuberculosis) or TST-negative individuals in South India, where TST positivity can define tuberculosis latency. RESULTS: Although purified protein derivative-specific and Mycobacterium tuberculosis culture filtrate antigen-specific Th1 and Th2 cytokines were not statistically significantly different between the 2 groups, the Th17 cytokines (interleukin 17 and interleukin 23) were statistically significantly decreased in TST-positive individuals, compared with those in TST-negative individuals. This Th17 cytokine modulation was associated with statistically significantly increased expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) and Foxp3. Although CTLA-4 blockade failed to restore full production of interleukin 17 and interleukin 23 in TST-positive individuals, depletion of regulatory T cells significantly increased production of these cytokines. CONCLUSION: TST positivity is characterized by increased activity of regulatory T cells and a coincident down-regulation of the Th17 response.

Impact of advocacy on the tuberculosis management practices of private practitioners in Chennai City, India.

BACKGROUND: Innovative schemes to ensure the participation of private practitioners (PPs) in the Revised National Tuberculosis Control Programme (RNTCP) are necessary to identify and treat all patients with tuberculosis (TB). We developed a novel public-private mix (PPM) model to encourage PPs to practise DOTS and participate in the RNTCP while retaining their patients. METHODS: The Resource Group for Education and Advocacy for Community Health (REACH) developed and implemented the programme in partnership with the Chennai local health authority and the Tuberculosis Research Centre, Chennai, India. PPs were sensitised to the RNTCP and DOTS through a one-to-one approach or group meetings, and were assisted in referring patients. Surveys were carried out at baseline and at the completion of the study to assess changes in attitudes and practices. RESULTS: Six hundred PPs underwent sensitisation about the RNTCP, after which the proportion of PPs adopting DOTS increased significantly (P < 0.001), and the majority (72.8%) used sputum testing for diagnosing TB. The proportion of PPs who used X-ray alone for diagnosis declined to 16.0% from a baseline of 45.4%. CONCLUSIONS: This PPM model, which emphasises sustained advocacy for DOTS and allows PPs to retain private patients, looks promising and needs to be tested at other sites.

Pharmacokinetics of lamivudine & stavudine in generic fixed-dose combinations in HIV-1 infected adults in India.

BACKGROUND & OBJECTIVES: Antiretroviral drug concentrations are important determinants of clinical response to a drug accounting for both toxicity and efficacy. Several factors such as age, ethnicity, body weight and patients' immune status may influence antiretroviral drug concentrations. The aim of the study was to determine the influence of immunological status, sex and body mass index on the steady state pharmacokinetics of lamivudine (3TC) and stavudine (d4T) in HIV-infected adults, who were undergoing treatment with generic fixed dose combinations (FDC) of these drugs in India. METHODS: Twenty seven HIV-1 infected patients receiving antiretroviral treatment (ART) for at least two weeks at the Government ART clinic at Tambaram, Chennai, took part in the study. Serial blood samples were collected predosing and at different time points after drug administration. Plasma 3TC and d4T levels were estimated by HPLC. RESULTS: The patients' immune status, sex or body mass index had no impact on the pharmacokinetics of 3TC. In the case of d4T, peak concentration was significantly lower in patients with CD4 cell counts < 200 cells/microl than those with > or = 200 cells/ microl (P < 0.05), but were within the therapeutic range. The mean CD4 cell counts increased from 101 cells/microl at initiation of ART to 366 cells/microl at 12 months of treatment. INTERPRETATION & CONCLUSIONS: Blood levels of 3TC and d4T drugs that are part of generic FDCs commonly used by HIV-infected individuals in India were within the therapeutic range and not influenced by nutritional or immune status. There was a significant improvement in CD4 cell counts over 12 months of treatment. Indian generic FDCs manufactured and used widely in the developing world provide effective concentrations of antiretroviral drugs.

Relevance of anti-BmR1 IgG4 antibodies in children from an area endemic for Brugia malayi infection in Kerala, India.

Brugian filariasis prevalent mostly in South-East Asian countries including India contributes to a small but significant proportion of the socioeconomic burden due to lymphatic filariasis. Along with bancroftian filariasis, brugian filariasis has been targeted for elimination globally. The lack of a reliable daytime diagnostic test has been seen as an important barrier to the successful implementation and monitoring of elimination programmes in brugia endemic areas. We evaluated an anti-BmRI-IgG4 antibody test namely, 'Brugia Rapid' in a large study meant to understand the clinical and pathological manifestations of brugian filariasis in children. We found the test superior to traditional night blood screening for microfilaraemia. Although an antibody detection test, we found it to be a reliable indicator of brugian infection. Among the 100 children studied extensively, 94% of the microfilaraemics, 86% of those showing filarial dance sign indicating presence of, live adult worms and 78% having abnormal lymphatics on lymphoscintigraphy were IgG4 positive. Coupled with its advantages like ease of use any time of the day, high sensitivity and specificity, this test may be the ideal tool to assist programme managers in their efforts to eliminate lymphatic filariasis where brugian infections are found.

Excess mortality and risk factors for mortality among a cohort of TB patients from rural south India.

OBJECTIVES: To estimate the excess general mortality among tuberculosis (TB) patients in a rural area (Tiruvallur) and identify risk factors for TB-related mortality. SETTING: The study population consisted of all TB patients aged >or=15 years who were registered under the Revised National Tuberculosis Control Programme (RNTCP) during the years 2000 to 2003 at Velliyur TB unit (TU) in south India. DESIGN: This is a retrospective cohort study of 3405 patients treated under the DOTS strategy, followed up from the date of start of treatment till the date of interview (for the survivors) or the date of death (for those who died). RESULTS: There were 2710 (79.6%) survivors and 695 (20.4%) deaths. The excess general mortalities for the cohort, expressed as standardised mortality ratio (SMR), was 4.2 (95%CI 3.9-4.5). High SMR values were obtained for patients belonging to the 15-44 years age group (12.1), patients on Category II regimen (9.3), treatment failures (9.1) and defaulters (7.8). The adjusted hazards ratios (aHR) were high for patients aged 45-59 years (1.9), >or=60 years (3.1) and with incomplete treatment due to default or failure (6.4). CONCLUSION: TB is one of the main causes of mortality in the younger age group. Among TB patients, the major risk factors for mortality are old age (>or=45 years) and incomplete treatment.

Lymphoscintigraphic evidence of lymph vessel dilation in the limbs of children with Brugia malayi infection.

Lymphatic filariasis (LF) is targeted for global elimination by the year 2020. It was earlier believed that LF is mostly a disease of adults. Recent studies indicate that in endemic countries filarial infection starts mostly in childhood even though the disease manifestations occur much later in life. The initial damage to the lymph vessels where the adult worms are lodged is dilation, thought to be irreversible even with treatment. Most of these studies relate to bancroftian filariasis. Studies that address this early pathology in brugian filariasis in humans are scarce. We report here for the first time, the lymphatic abnormalities seen on lymphoscintigraphy (LSG) in children with Brugia malayi filariasis. LSG was performed in 100 children aged between 3-15 years, who were enrolled in the study either because they were microfilaremic; had present or past filarial disease or were positive for antifilarial IgG4 antibodies. Inguinal and axillary lymph nodes were imaged in most children. Dilated lymph vessels were visualized in 80 children and this pathology was evenly distributed in all the three study groups. Lymph vessels dilation was seen even in three year old children. The implications of these findings for management of LF and control programmes are discussed.

Parasite-specific anergy in human filariasis. Insights after analysis of parasite antigen-driven lymphokine production.

The antigen-specific immune unresponsiveness seen in bancroftian filariasis was studied by examining lymphokine production in peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from 10 patients with asymptomatic microfilaremia, 13 patients with elephantiasis and 6 normal North Americans. In each group of patients, the kinetics of the lymphokine response and the response to mitogens and nonparasite antigens did not differ significantly. In marked contrast, when antigen-induced lymphokine production was examined, most patients with microfilaremia were unable to produce either interleukin 2 (IL-2) or gamma-interferon (i.e., were nonresponders), and the few who could (hyporesponders, generally with quite low microfilaremia levels) did so at levels significantly less than those of patients with elephantiasis, all of whom showed strong responses to parasite antigen. Removal of neither adherent cells or T8+ cells affected the parasite-specific anergy seen in those with microfilaremia, suggesting a state of T cell tolerance to the parasite in patients with this most common clinical manifestation of bancroftian filariasis.

Prenatal allergic sensitization to helminth antigens in offspring of parasite-infected mothers.

Total and filaria-specific immunoglobulin E (IgE) levels were studied in cord blood from infants born in Madras, India, where filariasis and intestinal helminth infections are highly endemic. Increased total IgE levels were observed in 82% of 57 cord sera tested (geometric mean 12.6 ng/ml; range 1-1,900 ng/ml). 33 of these sera also contained IgE antibodies specific for filarial antigens as determined by solid-phase radioimmunoassay. Comparison of ratios of filaria-specific IgE to total IgE in paired maternal and cord sera suggested that cord blood IgE was derived from the fetus in most cases and not from transplacental antibody transfer. Our results suggest that prenatal allergic sensitization to helminth parasites occurs in the tropics. Such sensitization may contribute to the heterogeneity in host immune response and disease expression noted in filariasis and other helminth infections.

Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset.

The immunological mechanisms involved in maintenance of an asymptomatic microfilaremic state (MF) in patients with lymphatic filariasis remain undefined. MF patients have impaired filarial antigen (Ag)-specific lymphocyte proliferation and decreased frequencies (Fo) of Ag-specific T cells, and yet elevated serum IgE and antifilarial IgG4. To investigate the mechanism of Ag-specific anergy in MF patients in contrast to amicrofilaremic individuals with chronic lymphatic obstruction (CP), the Fo of Ag-specific lymphocytes from peripheral blood mononuclear cells secreting either IL-4 or IFN-gamma were assessed by filter spot enzyme-linked immunosorbent assay, and IL-10 and transforming growth factor-beta (TGF-beta) mRNA transcript levels were assessed by a semiquantitative reverse transcriptase polymerase chain reaction technique. The Fo of filaria-specific IL-4-secreting lymphocytes were equivalent in both MF (geometric mean [GM] = 1:11,700) and CP (GM = 1:29,300 P = 0.08), whereas the Fo of IFN-gamma-secreting lymphocytes were lower in MF (GM = 1:39,300) than in CP (GM = 1:4,200, P < 0.01). When the ratio of IL-4/IFN-gamma (T helper type 2 [Th2]/Th1)-secreting cells was examined, MF subjects showed a predominant Th2 response (8:1) compared with a Th1 response in CP individuals (1:4). mRNA transcript levels of IL-10 were also significantly elevated in MF compared with CP individuals (P < 0.01). Further, IL-10 and TGF-beta were shown to have a role in modulating the Ag-specific anergy among MF subjects, in that neutralizing anti-IL-10 or anti-TGF-beta significantly enhanced lymphocyte proliferation response (by 220-1,300%) to filarial Ags in MF individuals. These findings demonstrate that MF subjects respond to parasite antigen by producing a set of suppressive cytokines that may facilitate persistence of the parasite within humans while producing little clinical disease.

Immunologic tolerance in lymphatic filariasis. Diminished parasite-specific T and B lymphocyte precursor frequency in the microfilaremic state.

To explore the mechanisms of antigen-specific immune unresponsiveness seen in microfilaremic patients with bancroftian filariasis, T and B cell precursor frequency analysis was performed using PBMC from individuals with either asymptomatic microfilaremia (MF, n = 7) or chronic lymphatic obstruction (CP, n = 20). Highly purified CD3+ cells were partially reconstituted with adherent cells and their proliferative response to parasite antigens determined in cultures of T cells by limiting dilution analysis. A filter immunoplaque assay also assessed the frequency of both total and parasite-specific Ig-producing B cells. While the lymphocyte proliferation to mitogens and to a nonparasite antigen (Streptolysin-O, [SLO]) were similar in all groups of patients, the frequency of parasite-specific CD3+ T cells was significantly lower (geometric mean [GM], 1/3,757) in MF patients when compared to that in CP patients (GM 1/1,513; P less than 0.001). Similarly, the proportion of lymphocytes producing parasite-specific IgE or IgG was significantly lower in MF patients (IgE mean, 0.2%; IgG mean, 0.33%) compared with CP patients (IgE mean, 3.2%; IgG mean, 1.76%; P less than 0.05 for both comparisons). These observations imply that low numbers of parasite-specific T and B lymphocytes may be partially responsible for the severely diminished capacity of lymphocytes from patients with MF to produce parasite-specific antibody and to proliferate to parasite antigen in vitro. Such differences in parasite-specific lymphocyte responses suggest that tolerance by clonal anergy may be a critical mechanism for maintaining the microfilaremic state.

Acute tropical pulmonary eosinophilia. Characterization of the lower respiratory tract inflammation and its response to therapy.

Although acute tropical pulmonary eosinophilia (TPE) is well recognized as a manifestation of filarial infection, the processes that mediate the abnormalities of the lung in TPE are unknown. To evaluate the hypothesis that the derangements of the lower respiratory tract in this disorder are mediated by inflammatory cells in the local milieu, we utilized bronchoalveolar lavage to evaluate affected individuals before and after therapy. Inflammatory cells recovered from the lower respiratory tract of individuals with acute, untreated TPE (n = 8) revealed a striking eosinophilic alveolitis, with marked elevations in both the proportion of eosinophils (TPE 54 +/- 5%; normal 2 +/- 5%; P less than 0.001) and the concentration of eosinophils in the recovered epithelial lining fluid (ELF) (TPE 63 +/- 20 X 10(3)/microliter; normal 0.3 +/- 0.1 X 10(3)/microliter; P less than 0.01). Importantly, when individuals (n = 5) with acute TPE were treated with diethylcarbamazine (DEC), there was a marked decrease of the lung eosinophils and concomitant increase in lung function. These observations are consistent with the concept that at least some of the abnormalities found in the lung in acute TPE are mediated by an eosinophil-dominated inflammatory process in the lower respiratory tract.

Urine levels of rifampicin & isoniazid in asymptomatic HIV-positive individuals.

BACKGROUND & OBJECTIVE: AIDS and its associated gastrointestinal complications may impair the absorption of anti-tuberculosis (TB) drugs. Impaired absorption of anti-TB drugs could lead to low drug exposure, which might contribute to acquired drug resistance and reduced effectiveness of anti-TB treatment. The aim of this study was to obtain information on the status of absorption of rifampicin (RMP) and isoniazid (INH) in asymptomatic HIV- positive individuals, who are less immunocompromised. The D-xylose absorption test was also carried out to assess the absorptive capacity of intestive. METHODS: The absorption of RMP, INH and D-xylose was studied in 15 asymptomatic HIV-positive individuals with CD4 cell counts>350 cells/mm3 and 16 healthy volunteers, after oral administration of single doses of RMP (450 mg), INH (300 mg) and D-xylose (5 g). Urine was collected up to 8 h after drug administration. Percentage dose of the drugs and their metabolites and D-xylose excreted in urine were calculated. RESULTS: A significant reduction in the urinary excretion of INH and D-xylose in HIV-positive persons compared to healthy volunteers was observed. The per cent dose of RMP and its metabolite, desacetyl RMP was also lower in HIV-positive persons compared to healthy volunteers, but this difference was not statistically significant. INTERPRETATION & CONCLUSION: Decreased urinary excretion of D-xylose and INH are suggestive of intestinal malabsorption in HIV-positive individuals. HIV infection could cause malabsorption of anti-TB drugs even at an early stage of the disease. The clinical implications of these findings need to be confirmed in larger studies.

Coincident filarial, intestinal helminth, and mycobacterial infection: helminths fail to influence tuberculin reactivity, but BCG influences hookworm prevalence.

The prevalence of helminth and tuberculosis infections is high in South India, whereas Bacille-Calmette-Guerin (BCG) vaccine efficacy is low. Our aim was to determine whether concurrent helminth infection alters the ability to mount a delayed-type hypersensitivity response to tuberculin. In a cross-sectional study in southern India, individuals 6-65 years of age were screened for intestinal helminths, circulating filarial antigenemia, tuberculin reactivity, active tuberculosis, and history of BCG vaccination; 54% were purified protein derivative (PPD) positive, 32% had intestinal helminth infection, 9% were circulating filarial antigen positive, and 0.5% had culture-confirmed active tuberculosis. Only age and BCG vaccination were significantly associated with PPD reactivity; however, BCG vaccination was associated with a lower prevalence of hookworm infection relative to those without prior BCG vaccination. Neither intestinal helminth infection nor filarial infection was associated with diminished frequencies of PPD positivity. Our findings suggest that preceding helminth infection does not influence significantly the delayed-type hypersensitivity response to tuberculin.

Relationship of ARTI to incidence and prevalence of tuberculosis in a district of south India.

OBJECTIVE: To validate the currently used empirical relationship between annual risk of tuberculous infection (ARTI) and incidence and prevalence of smear-positive cases. SETTING: Two disease surveys to estimate the prevalence and incidence of tuberculosis (TB) among adults in Tiruvallur district, south India, and a tuberculin survey to estimate the ARTI among children. RESULTS: The incidence of TB was estimated to be 82 and prevalence 210 per 100,000 population and ARTI 1.6%. We estimated that 1% ARTI corresponded to 51 new and 131 prevalent cases. CONCLUSION: The currently used empirical relationship between ARTI and incidence can be used by programme managers as an effective monitoring tool.

A simple and rapid liquid chromatography method for simultaneous determination of zidovudine and nevirapine in plasma.

We describe a simple, fast, isocratic, reversed-phase high performance liquid chromatographic method for simultaneous determination of plasma zidovudine and nevirapine with UV detection at 260 nm. The method involves liquid-liquid extraction with ethyl acetate and using 3-isobutyl 1-methyl xanthine as internal standard. The system requires a C(18) column (150 mm x 4.6 mm I.D.) and a mobile phase composed of potassium dihydrogen phosphate (15 mM; pH 7.5) and acetonitrile in the ratio of 80:20 (v/v). The assay was linear from 0.025 to 10.0 microg/ml for zidovudine and 0.05 to 10.0 microg/ml for nevirapine. The intra- and inter-day variations were less than 10% for both the drugs. The method was specific and sensitive enough to allow quantification of zidovudine and nevirapine in concentrations observed clinically. The average recoveries of zidovudine and nevirapine from plasma were 95 and 94%, respectively. The method was applied to a pharmacokinetic study in HIV-infected patients who were receiving antiretroviral treatment with zidovudine and nevirapine containing regimens. The method spans the blood concentration range of clinical interest. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring in patients taking a combination treatment of zidovudine and nevirapine.