Distribution and spread of pyrethroid and DDT resistance among the Anopheles gambiae complex in Tanzania.

The development of insecticide resistance is a threat to the control of malaria in Africa. We report the findings of a national survey carried out in Tanzania in 2011 to monitor the susceptibility of malaria vectors to pyrethroid, organophosphate, carbamate and DDT insecticides, and compare these findings with those identified in 2004 and 2010. Standard World Health Organization (WHO) methods were used to detect knock-down and mortality rates in wild female Anopheles gambiae s.l. (Diptera: Culicidae) collected from 14 sentinel districts. Diagnostic doses of the pyrethroids deltamethrin, lambdacyhalothrin and permethrin, the carbamate propoxur, the organophosphate fenitrothion and the organochlorine DDT were used. Anopheles gambiae s.l. was resistant to permethrin in Muleba, where a mortality rate of 11% [95% confidence interval (CI) 6-19%] was recorded, Muheza (mortality rate of 75%, 95% CI 66-83%), Moshi and Arumeru (mortality rates of 74% in both). Similarly, resistance was reported to lambdacyhalothrin in Muleba, Muheza, Moshi and Arumeru (mortality rates of 31-82%), and to deltamethrin in Muleba, Moshi and Muheza (mortality rates of 28-75%). Resistance to DDT was reported in Muleba. No resistance to the carbamate propoxur or the organophosphate fenitrothion was observed. Anopheles gambiae s.l. is becoming resistant to pyrethoids and DDT in several parts of Tanzania. This has coincided with the scaling up of vector control measures. Resistance may impair the effectiveness of these interventions and therefore demands close monitoring and the adoption of a resistance management strategy.

Outbreak of tuberculosis in a homeless population involving multiple sites of transmission.

SETTING: During 2002-2003, a large outbreak of tuberculosis (TB) occurred among persons using multiple homeless facilities in King County, Washington. OBJECTIVE: To control the transmission of TB in multiple settings. DESIGN: In 2002, contacts exposed to patients in homeless facilities were screened using tuberculin skin tests (TSTs) and symptom review. Based on these screening results, sites of transmission were identified and prioritised, and exposed cohorts at these sites were offered intensive screening tests in 2003 (e.g., symptom review, TST, chest radiograph [CXR], sputum examination and culture). Mycobacterium tuberculosis isolates from patients were genotyped using PCR-based methods to identify outbreak-associated patients quickly. RESULTS: During 2002-2003, 48 (15%) of 313 patients diagnosed in King County were outbreak-associated; 47 culture-positive patients had isolates that matched the outbreak strain by genotyping. Three facilities visited by >12 patients in 2002 had a higher prevalence of TST positive results (approximately 30%) among clients compared with the background rate (7%) in the homeless community. Screening contacts with one sputum culture was as sensitive as CXR in detecting TB disease (77% vs. 62%, respectively). CONCLUSIONS: A comprehensive, resource-intensive approach likely helped to control transmission. This outbreak highlights the vulnerability of homeless populations and the need to maintain robust TB programs in urban settings.

Contact tracing: comparing the approaches for sexually transmitted diseases and tuberculosis.

SETTING: Literature review for the process of contact tracing for sexually transmitted diseases (STD) and for tuberculosis (TB), focusing on articles that report results of studies or commentary. OBJECTIVE: To compare and contrast contact tracing in order to highlight emerging commonalities. DESIGN: A descriptive review, based on Medline search with augmentation from other published and unpublished sources. RESULTS: Contact tracing for STD and TB have some obvious differences because of differing routes of transmission, differing sensibilities required to work with the affected populations, a different potential for anonymous contacts, and a major difference in the epidemiologic value of biomarkers. Nonetheless, the convergence of these processes on disadvantaged populations where drug use and sexual activity are important social factors has engendered an increasing similarity. CONCLUSION: A broadened approach to both, with greater attention to how ancillary contacts and associates may be of use in interrupting deeply embedded endemic disease transmission, deserves further study. Some newer approaches in the use of network-informed methods to elicit contacts and investigate the community dynamics of transmission may be of particular value in TB case investigation. These strategies will be enhanced by the availability of DNA fingerprinting, a powerful biomarker of recent Mycobacterium tuberculosis transmission and case association (a technology not available for STD contact tracing).

A network-informed approach to investigating a tuberculosis outbreak: implications for enhancing contact investigations.

BACKGROUND: To elucidate networks of Mycobacterium tuberculosis transmission, it may be appropriate to characterize the types of relationships among tuberculosis (TB) cases and their contacts (with and without latent TB infection) in addition to relying on traditional efforts to distinguish 'close' from 'casual' contacts. SETTING: A TB outbreak in a US low incidence state. OBJECTIVE: To evaluate whether social network analysis can provide insights into transmission settings that might otherwise go unrecognized by routine practices. DESIGN: All adult outbreak-associated cases (n = 19) and a convenience sample of their contacts with and without latent TB infection (LTBI) (n = 26) were re-interviewed in 2001 using a structured questionnaire. Network analysis software was used to create diagrams illustrating important persons within the outbreak network, as well as types of activities TB cases engaged in with their contacts. RESULTS: Drug use and drug sharing were more commonly reported among cases and their infected contacts than among contacts without LTBI. TB cases central to the outbreak network used crack cocaine, uncovering the need to focus control efforts on specific sites and persons involved in illicit drug use. CONCLUSION: Outbreaks occur even in areas with low TB incidence, frequently among groups whose drug use or other illegal activities complicate control efforts. TB programs should consider the use of network analysis as a supplement to routine contact investigations to identify unrecognized patterns of M. tuberculosis transmission.

Tumor necrosis factor-alpha promoter variant 2 (TNF2) is associated with pre-term delivery, infant mortality, and malaria morbidity in western Kenya: Asembo Bay Cohort Project IX.

A polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-alpha) gene, with a guanine to adenine nucleotide change at position -308, TNF2 is associated with increased TNF-alpha production. TNF2 homozygotes have a higher risk of severe disease and/or death due to cerebral malaria and other infectious diseases. We investigated the impact of this allele on malaria morbidity and mortality in young children who participated in an immuno-epidemiologic cohort study of malaria in an area of intense perennial Plasmodium falciparum transmission in western Kenya. A total of 1,048 children were genotyped. Poisson regression and Cox proportional hazards models were used to determine the relationship between TNF-308 variants and morbidity and mortality. The gene frequencies of the TNF1 and TNF2 alleles were 0.90 and 0.10, respectively. TNF2 homozygosity was associated with pre-term birth when compared with TNF1 homozygotes [relative risk (RR) 7.3, 95% CI, 2.85-18.9, P = 0.002) and heterozygotes (RR 6.7, 95% CI 2.0-23.0, P = 0.008). Among children born prematurely, the TNF2 allele was significantly associated with a higher risk of death in infancy compared with TNF1 (RR 7.47, 95% CI 2.36-23.6). The risk of death was higher among TNF2 homozygotes than among heterozygotes. The TNF2 allele was significantly associated with high density P. falciparum parasitemia (RR 1.11, 95% CI 1.0-1.24). Among low birth weight children, the TNF2 allele was associated with severe anemia (RR 2.16, 95% CI 1.17-4.01) and showed a trend toward a risk for severe malaria anemia (RR 1.99, 95% CI 0.89-4.46). These data suggest that TNF2 is a risk factor for pre-term birth and early childhood mortality and malaria morbidity in children in this region. Further understanding of the pathogenic mechanisms underlying this association is required.

Fcgamma receptor IIa (CD32) polymorphism is associated with protection of infants against high-density Plasmodium falciparum infection. VII. Asembo Bay Cohort Project.

In vitro studies have shown that inhibition of Plasmodium falciparum blood-stage parasite growth by antibody-dependent cellular inhibition is mediated by cooperation between malaria-specific IgG1 and IgG3, but not IgG2, and monocytes via the Fcgamma receptor II (FcgammaRII). A single amino acid substitution at position 131 in FcgammaRIIa is critical in the binding of human IgG subclasses. The hypothesis that the FcgammaRIIa-Arg/Arg131 genotype, which does not bind to IgG2, is a host genetic factor for protection against high-density P. falciparum infection was tested. One hundred eighty-two infants from a large community-based birth cohort study in western Kenya were selected for an unmatched case-control study. Results showed that the infants with the FcgammaRIIa-Arg/Arg131 genotype were significantly less likely to be at risk for high-density falciparum infection, compared with infants with the FcgammaRIIa-His/Arg131 genotype (adjusted odds ratio, 0.278; 95% confidence interval, 0.123-0.627; P=.0021). This finding supports the hypothesis.

All-cause mortality among young children in western Kenya. VI: the Asembo Bay Cohort Project.

Although all-cause mortality has been used as an indicator of the health status of childhood populations, such data are sparse for most rural areas of sub-Saharan Africa, particularly community-based estimates of infant mortality rates. The longitudinal follow-up of more than 1,500 children enrolled at birth into the Asembo Bay Cohort Project (ABCP) in western Kenya between 1992 and 1996 has provided a fixed birth cohort for estimating all-cause mortality over the first 5 yr of life. We surveyed mothers and guardians of cohort children in early 1999 to determine survival status. A total of 1,260 households were surveyed to determine the survival status of 1,556 live births (99.2% of original cohort, n = 1,570). Most mothers (66%) still resided but 27.5% had migrated, and 5.5% had died. In early 1999, the overall cumulative incidence of all-cause mortality for the entire 1992-1996 birth cohort was 26.5% (95% confidence interval, 24.1-28.9%). Neonatal and infant mortality were 32 and 176 per 1,000 live births, respectively. These community-based estimates of mortality in the ABCP area are substantially higher than for Kenya overall (nationally, infant mortality is 75 per 1,000 live births). The results provide a baseline description of all-cause mortality among children in an area with intense Plasmodium falciparum transmission and will be useful in future efforts to monitor changes in death rates attributable to control programs for specific diseases (e.g., malaria and HIV/AIDS) in Africa.

A multi-state outbreak of tuberculosis among members of a highly mobile social network: implications for tuberculosis elimination.

SETTING: Baltimore, Maryland. OBJECTIVE: To describe a tuberculosis (TB) outbreak among a highly mobile population and the efforts required to control it. DESIGN: Epidemiologic outbreak investigation. RESULTS: Between June 1998 and January 2000, 20 TB outbreak cases were identified, of which 18 were culture-confirmed. Seventeen isolates of Mycobacterium tuberculosis had an identical 11-band DNA fingerprint; another isolate had one additional band and was considered a match. Two cases were diagnosed in New York City; another patient lived primarily in Atlanta, but was diagnosed in Baltimore. Persons in the outbreak were predominantly young (median age 24 years), black, male, infected with the human immunodeficiency virus (HIV), and gay, transvestite or transsexual. Activities common among many TB cases included attending two nightclubs, membership in one of three social 'Houses', attending balls or pageants in East Coast cities, marijuana use, and prostitution. Community outreach, extended contact tracing, DNA fingerprinting, directly-observed therapy, and expanded use of preventive therapy were utilized to assess and control the outbreak. During the outbreak period the Baltimore City TB rate declined by 10%. However, additional public health personnel were required to control the outbreak, resulting in a 17% increase in TB clinic staff. CONCLUSION: As TB rates decline, remaining cases are likely to occur in difficult-to-reach populations. Increased resources per case of TB treated will be required to eliminate TB.

Effect of Plasmodium falciparum parasitemia density on hemoglobin concentrations among full-term, normal birth weight children in western Kenya, IV. The Asembo Bay Cohort Project.

The relative importance of acute high-density versus persistent low-density Plasmodium falciparum parasitemia in contributing to the public health problem of malarial anemia remains unclear. The Asembo Bay Cohort Project in western Kenya collected monthly hemoglobin (Hb) and parasitologic measurements and biweekly assessments of antimalarial drug use among 942 singleton live births between 1992 and 1996. A mixed-model analysis appropriate for repeated measures data was used to study how time-varying parasitemia and antimalarial drug exposures influenced mean Hb profiles. Incidence of World Health Organization-defined severe malarial anemia was 28.1 per 1,000 person-years. Among children aged less than 24 months, concurrent parasitemia was significantly associated with lower mean Hb, especially when compared to children with no concurrent parasitemia. Increased densities of the 90-day history of parasitemia preceding Hb measurement was more strongly associated with mean Hb levels than concurrent parasitemia density. While the highest quartile of 90-day parasitemia history was associated with lowest mean Hb levels, children in the lowest 90-day exposure quartile still experienced significantly lower Hb levels when compared to children who remained parasitemia-free for the same 90-day period. The results highlight the importance of collecting and analyzing longitudinal Hb and parasitologic data when studying the natural history of malarial anemia.

Analysis of repeated hemoglobin measures in full-term, normal birth weight Kenyan children between birth and four years of age. III. The Asemobo Bay Cohort Project.

Anemia is an important public health problem. During very early childhood numerous factors affect hemoglobin (Hb) concentration over time, making single cross-sectional measurements difficult to interpret when studying the natural history of anemia or evaluating anemia control strategies. We analyzed repeated Hb measures contributed by 942 Kenyan children between birth and 48 months of life using a mixed effects model, with a regression spline used to describe the population mean Hb profile, and random intercepts and slopes and first-order autoregressive correlation structure to accommodate the within-individual correlation among the repeated Hb measures. The approach facilitates the study of time-stationary and time-varying covariates that influence Hb in early life. The fitted mean Hb profile obtained from the analytic model is consistent with the observed mean Hb of the study population. Village of residence was associated with greatest difference in mean Hb at time of birth (16 versus 19 g/dL; P < 0.0001). Monthly weight-for-age was also associated with mean Hb after 3 months of age. This is the first description of an analysis strategy specifically for repeated Hb measures collected in a longitudinal field study in Africa. The strategy will facilitate improved study of time-varying covariates thought to influence pediatric anemia.

Dose- and time-dependent relations between infective Anopheles inoculation and outcomes of Plasmodium falciparum parasitemia among children in western Kenya.

Blood-stage level Plasmodium falciparum infection (parasitemia density) is generally elevated prior to, or at the time of, clinical presentation of severe pediatric malaria episodes. Intensity of exposure to infective Anopheles mosquito bites is a suspected determinant of higher density parasitemia. Analyses of entomologic and parasitologic data collected in 1986-1987 were conducted to investigate whether the dose of infective bites predicted the incidence or degree of P. falciparum parasitemia in Kenyan children < 6 years old. At 21 consecutive 30-day intervals, a new cohort (n approximately 50 each) was enrolled, cured of malaria parasites, and monitored over 84 days for recurrent parasitemia. Outcomes included time to parasitemia, time to parasitemia > or = 5,000/microliter, and parasitemia density. Ecologic and individual-level analyses were conducted. The mean infective bite exposure experienced by each cohort was significantly associated with the incidence of parasitemia (age-adjusted r2 = 0.38, p = 0.022) and more strongly associated with the incidence of parasitemia > or = 5,000/microliter (age-adjusted r2 = 0.72, p < 0.001). The infective bite dose, analyzed as a time-dependent covariate, was associated with a 2.8 times higher rate of parasitemia > or = 5,000/microliter among children exposed to > or = 1 infective bite per day as compared with the referent (rate ratio (RR) = 2.82, 95% confidence interval (CI) 2.24-3.56). Cumulative infective bite exposure, exposure duration, and age were significant predictors of recurrent parasitemia density in multiple linear regression analyses. The results support the contention that reductions in P. falciparum transmission intensity, in the absence of complete elimination, will reduce higher level parasitemia among African children.

PIP: Elevated numbers of asexual erythrocytic-stage Plasmodium falciparum parasites in the peripheral blood circulation is a known risk factor of the clinical severity of malaria episodes. The interrelationships among a continuum of sporozoite dose, duration of exposure, age, level of parasitemia at enrollment, village of residence, sex, and recurrent P. falciparum parasitemia were investigated in a 2-year (1986-87) study of 862 children 6 months to 6 years of age from six contiguous villages in Western Kenya. At 21 consecutive 30-day intervals, a new cohort was enrolled, cured, and monitored over 84 days for recurrent parasitemia. The mean cumulative dose was 23 inoculations, and there was a significant linear correlation between this variable and the incidence rate of first recurrent parasitemia, with even stronger associations for the incidence of higher density parasitemia. The overall 70-day cumulative incidence of first recurrent parasitemia was 88.5% (22.5% for high-density P. falciparum). The infective bite dose, analyzed as a time-dependent covariate, was associated with a 2.8 times higher rate of parasitemia equal to or above 5000/mcl among children exposed to one or more bites per day compared to the referent. Each one unit increase in the mean dose was associated with a 24% higher rate of recurrent parasitemia and a 26% higher rate of recurrent high-density parasitemia after adjustment for covariates. Multiple linear regression analyses indicated that parasitemia density was significantly positively associated with cumulative dose and inversely associated with duration of exposure and age. Approximately 36% of the variance in malaria incidence rates was explained by the mean cumulative dose of infective bites.

Plasmodium falciparum gametocytemia in Kenyan children: associations among age, intensity of exposure to transmission, and prevalence and density of subsequent gametocytemia.

Recently, an association was described between the density of Plasmodium falciparum asexual parasitemia in Kenyan children and the entomologic inoculation rate (EIR) measured prior to measurement of asexual parasitemia. This study examined whether transmission pressure, as represented by the EIR, was associated with the prevalence or density of gametocytemia in Kenyan children. Each month for 19 months, a cohort of approximately 50 children was given a radical cure and enrolled in the study. Blood films were taken on days 0, 7, and 14. The EIR was calculated for the 28-day period ending 14 days prior to enrollment: the relationship between blood film data from day 7 and exposure variables was explored. We found that younger children were more likely to be gametocytemic than older children and, if gametocytemic, were more likely to have a dense gametocytemia. There was an inverse relationship between the number of infective bites per night received and prevalence but not density of gametocytemia, even after age adjustment. Concordance of gametocytemia prevalence on days 0 (64%), 7 (66%), and 14 (52%) was poor; 84% of the children were positive on at least one day. This indicates that in many subjects the detectable gametocytemia varied over the 14 days. Under these holoendemic transmission conditions, the EIR is inversely correlated with prevalence of gametocytemia, and point measurements of gametocytemia by conventional microscopy underestimate the number of infective donor hosts.

Impact of transmission intensity and age on Plasmodium falciparum density and associated fever: implications for malaria vaccine trial design.

To facilitate design of vaccine trials, malaria was studied in 6-month- to 6-year-old Kenyans during high (HI) and low intensity transmission seasons. During 84 days after cure, exposure to infected mosquitoes was 9-fold greater in the HI group, yet incidence of P. falciparum infection was increased only 2-fold, with no age effect. The density of recurrent P. falciparum was 14-fold greater in the HI group, and there was a striking association between age and parasitemia > or = 5000/microL. Fever was the only clinical manifestation attributable to parasitemia and only when the parasite density was > or = 5000/microL. Sixty-four percent of children with > or = 20,000 parasites/microL versus 10% with 1-4999/microL were febrile when parasitemic. Recurrent P. falciparum infection as a vaccine trial end point can be studied year-round among children < or = 6 years [corrected] in western Kenya. However, high-grade parasitemia (> or = 5000 or 20,000/microL) with or without elevated temperature will be optimally studied in the high transmission season among children < 2 years.

Treatment of chloroquine-resistant Plasmodium vivax with chloroquine and primaquine or halofantrine.

Optimal therapy for infection by chloroquine-resistant Plasmodium vivax has not been established. From 1992 to 1994 during three separate studies, 147 Javanese residents of Irian Jaya infected by P. vivax were treated with either chloroquine (25 mg of base/kg during 3 days or 10 mg of base/kg in one dose) plus primaquine (10 mg/kg during 28 days or 2.5 mg/kg during 3 days) (n = 78), chloroquine plus placebo (n = 50), or halofantrine (24 mg base/kg in 12 h; n = 19). There was no difference in tolerance to or side effects of any of the regimens. Within 14 days of starting therapy, therapeutic failure among these patients was 44% for chloroquine, 5% for chloroquine plus primaquine (P < .001), and 0 for halofantrine (P < .001). After 28 days, therapeutic failure was 78%, 15%, and 6%, respectively. Thus, chloroquine plus primaquine in combination and halofantrine alone are effective therapies for chloroquine-resistant P. vivax.

Predicting outcome in malaria: correlation between rate of exposure to infected mosquitoes and level of Plasmodium falciparum parasitemia.

The level of Plasmodium falciparum parasitemia at clinical presentation has repeatedly been shown to correlate with severity of disease. Using data collected in western Kenya over 21 months, we examined associations between exposure variables, especially exposure to infective mosquitoes, and prevalence and density of P. falciparum parasitemia among 1,007 children six months to six years of age. The prevalence of P falciparum infection was similar at all exposure levels, but there was a correlation between exposure to sporozoite-infected mosquitoes over the previous 28-day period, and geometric mean parasite density of each cohort (Spearman rank coefficient = 0.724, P = 0.002). The relative odds of having a parasite density > or = 5,000/microliters was increased almost two-fold among individuals exposed to more than 10 infective bites during the prior 28-day period. Children enrolled during the highest incidence period were 80% more likely to have a density > or = 5,000/microliters relative to individuals enrolled during periods of lower incidence. The data suggest that measures, such as malaria vaccines, that reduce parasite densities by limiting numbers of sporozoites reaching the liver, or merozoites released from the liver, will reduce malaria-associated morbidity and mortality, even when they do not prevent all infections.

Diagnosis of malaria by detection of Plasmodium falciparum HRP-2 antigen with a rapid dipstick antigen-capture assay.

Two field studies in Kenya and an experimental challenge study in the USA were done to assess the accuracy of a dipstick antigen-capture assay based on qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) in peripheral blood for diagnosis of P falciparum infection. In these studies, the assay was 96.5-100% sensitive for detection of greater than 60 P falciparum asexual parasites/microL blood, 70-81% sensitive for 11-60 parasites/microL blood, and 11-67% sensitive for 10 parasites or less/microL blood. Specificity was 95% (95% CI 85-105%; n = 20) among naive American volunteers, 98% (96-101%; n = 112) among volunteers exposed to the bite of P falciparum-infected mosquitoes, and 88% (84-92%; n = 285) among Kenyans living in an area with holoendemic malaria. Our results also indicated that PfHRP-2 antigen was not detectable in blood 6 days after initiation of curative chemotherapy, and suggest that such circulating antigens rarely lead to false-positive tests. The dipstick assay's sensitivity, specificity, simplicity, and speed may make it an important tool in the battle against malaria.

Malaria vaccine study site in Irian Jaya, Indonesia: Plasmodium falciparum incidence measurements and epidemiologic considerations in sample size estimation.

Malaria epidemiologic and entomologic studies were performed during both the high transmission and low transmission seasons to characterize the Plasmodium falciparum malaria transmission at a proposed malaria vaccine trial site in Irian Jaya, Indonesia. The study population consisted of two subsets: native Irianese men with lifelong exposure to malaria and transmigrants who arrived from a nonmalarious area 2.5 years before the start of the study. All subjects received a radical cure for malaria and were then monitored weekly by blood film. Both P. falciparum malaria attack rates and incidence densities were calculated; transmigrants had a significantly higher rate (P = 0.003) than the Irianese during the low transmission season study (20-weeks long) but not during the high transmission season study (12-weeks long). Lack of exposure-induced immunity left the transmigrants at a minimum 17-25% greater relative risk of becoming parasitemic compared with the Irianese during the low transmission season study. During the high transmission season study, 50% of the transmigrants were P. falciparum positive by week 6 and 50% of the Irianese by week 9. During the low transmission season, 50% of the transmigrants were positive by week 10 and 43% of the Irianese were positive by week 17. Entomologic studies showed that Anopheles koliensis was the predominant vector (> 98% of anopheline catch). Entomologic inoculation rates for P. falciparum were 0.018 and 0.39 infective bites/person/night for the low and high transmission seasons, respectively. New P. vivax cases represented between 16% and 42% of all initial malaria cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of current exposure to environmental tobacco smoke.

Reports of recent exposure to environmental tobacco smoke (ETS) and urinary cotinine levels were obtained on 663 never- and ex-smokers who attended a cancer screening clinic in Buffalo, New York, in 1986. Study objectives included determining the prevalence of exposure to ETS using urinary cotinine and identifying questionnaire exposure measures predictive of cotinine. Findings demonstrate that exposure to environmental tobacco smoke is extremely prevalent, even among those not living with a smoker. A total of 76% of subjects reported exposure to ETS in the 4 d preceding the interview. The most frequently mentioned sources of exposure were at work (28%) and at home (27%). Cotinine was found in the urine of 91% of subjects. Cotinine values increased significantly with the number of exposures reported. Among the different questionnaire measures of exposure that were evaluated, the single best predictor of cotinine was the number of friends and family members seen regularly by the subject who smoke.