Risk factors associated with typhoid fever in children aged 2-16 years in Karachi, Pakistan.

We analysed the data from the control group in a typhoid vaccine trial in Karachi to assess the differences in individual-, household- and cluster-level characteristics for developing typhoid fever. The annual incidence of typhoid in children aged 2-16 years in the control arm of the vaccine trial was 151/100 000 population. After adjustment, the risk of typhoid was lower with increasing age [risk ratio (RR) 0·89, 95% confidence interval (CI) 0·83-0·95], was higher with an increase in population density (RR 1·13, 95% CI 1·05-1·21) and was lower in the households using a safe drinking-water source (RR 0·63, 95% CI 0·41-0·99). Typhoid fever affects younger children living in areas of high population density and lack of access to safe water in Pakistan. A combination of environmental and biological interventions is required to prevent the continued epidemiological and economic impact of typhoid fever in high-risk areas of Pakistan.

Community perceptions of bloody diarrhoea in an urban slum in South Asia: implications for introduction of a Shigella vaccine.

Understanding local perceptions of disease causation could help public health officials improve strategies to prevent bloody diarrhoea. A cross-sectional survey was conducted in Dhaka, Bangladesh to elicit community beliefs about the causes of and prevention strategies for bloody diarrhoea. Between March and June 2003, we interviewed 541 randomly selected respondents. Overall, 507 (93%) respondents perceived that a vaccine could prevent bloody diarrhoea. If a vaccine provided lifetime protection, 445 (83%) respondents stated that they would opt to get the vaccine and would pay a median of $0·05 (range U.S.$0·01-0·15) for it, equivalent to <1% of their median weekly income. There was almost universal perception that an effective vaccine to prevent bloody diarrhoea was highly beneficial and acceptable. While respondents valued a vaccine for prevention of bloody diarrhoea, they were only willing to pay minimally for it. Therefore, achieving a high rate of Shigella vaccine coverage may require subsidy of vaccine purchase.

Non-typhoidal Salmonella rates in febrile children at sites in five Asian countries.

There is increased recognition of non-typhoidal Salmonella (NTS) as a major cause of severe febrile illness in sub-Saharan Africa. However, little is known about community-based incidence of NTS in Asia. In a multicentre, community-based prospective Salmonella surveillance study, we identified a total of six NTS cases: three in Karachi, Pakistan, one in Kolkata, India, and two in North Jakarta, Indonesia. No NTS cases were identified in Hechi, People's Republic of China, and Hue, Viet Nam. Three cases were in children under 3 years, and one case was in a child aged 10 years and one in a child aged 15 years. Only one case was an adult (29 years). The highest incidence of NTS infection was in Karachi (7.2 culture-proven NTS cases per 100,000 person years in age group of 2-15 years). However, in comparison with sub-Saharan Africa, the NTS burden in Asia appears rather limited.

The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin.

Although chloroquine-resistance (CQR) in Plasmodium falciparum is increasing and resistance to other blood schizonticidal anti-malarials has been reported, the molecular basis remains unclear. In this study fresh field isolates were obtained from The Gambia, an area of emerging CQR and tested for sensitivity to the anti-malarial drugs mefloquine, halofantrine, artemisinin, dihydroartemisinin, chloroquine and quinine. Sequence polymorphisms in the pfmdr1 gene and size polymorphisms in the cg2 gene were assessed using PCR-based systems. A strong association was observed between the presence of the tyr-86 allele of pfmdr1 and increased sensitivity to mefloquine and halofantrine, as well as the structurally unrelated drugs artemisinin and dihydroartemisinin. A weaker association was found between the presence of tyr-86 and increased resistance to chloroquine and quinine. The cg2 Dd2-like omega repeat size polymorphism was associated with increased resistance to chloroquine and increased sensitivity to mefloquine and halofantrine. An intragenic association was also found between a polymorphism in the polyasparagine linker region of pfmdr1 and the tyr-86 allele, which may be due to genetic hitchhiking, indicative of recent selection by chloroquine. Our data support a hypothesis where the pfmdr1 gene confers a true multidrug resistance phenotype which is lost by mutation.

Risk factors for gametocyte carriage in Gambian children.

A widespread reduction in Plasmodium falciparum gametocyte prevalence could reduce malaria transmission. After infection with P. falciparum, a variable proportion of people are found to be gametocytemic. We analyzed risk factors associated with gametocytemia at presentation and 7 days later. We enrolled 1,198 children in 2 antimalarial drug trials between September and December 1998. The children were assigned to 1 of 4 treatment groups: chloroquine only; pyrimethamine-sulfadoxine (PSD) only; PSD combined with 1 dose of artesunate; and PSD combined with 3 doses of artesunate. By the time of enrollment, 200 (17%) of 1,198 children were gametocyte carriers. Three independent risk factors were associated with gametocytemia at enrollment. Children with anemia were more likely to carry gametocytes, whereas children with fever (> 37.4 degrees C) or high parasite densities (> 100,000 parasites/microL) were less frequently gametocyte carriers. Children with at least 2 of the risk factors were 4 times more likely to be gametocytemic than children with < 2 risk factors (odds ratio [OR], 4.4; 95% confidence interval [CI], 2.7-7.1). Seven days after the start of treatment, 355 (37%) of 466 assessable children were found to be gametocyte carriers. Children treated with PSD alone had a significantly higher risk of being gametocytemic by Day 7 compared with children in the other 3 treatment groups. In the subgroup of children who had no detectable gametocytes on enrollment, the effect of treatment with PSD + 3 doses of artesunate was most marked. Nineteen (10%) of 198 children treated with PSD + 3 doses of artesunate became gametocytemic, in contrast to 184 (57%) of 321 children treated with PSD alone (OR, 12.7; 95% CI, 7.3-22.1). Early treatment with highly effective antimalarial therapy has the greatest chance of preventing gametocytemia. The choice of a first-line antimalarial drug for uncomplicated malaria should not only take into consideration the ablation asexual parasitemia but also the suppression of gametocytemia.

A randomized controlled trial of artemether/benflumetol, a new antimalarial and pyrimethamine/sulfadoxine in the treatment of uncomplicated falciparum malaria in African children.

We report here the results of a randomized double blind trial comparing coartemether (CGP56697), a combination of artemether and benflumetol, with pyrimethamine/sulfadoxine (P/S). Two hundred eighty-seven children 1-5 years of age with uncomplicated falciparum malaria were enrolled at two centers in The Gambia between July 1996 and December 1996. Following treatment, children were visited at home every 24 hr until a blood film free of asexual parasites was obtained. Genotyping of parasites was used to distinguish recrudescence from new infections. Three days after the start of treatment, 133 (100%) of the CGP56697-treated children compared with 128 (93.4%) of children treated with P/S had cleared their parasites (P = 0.003). The day 15 cure rate was 93.3% for CGP56697 and 97.7% for P/S (P = 0.13). Within the third and fourth week after initiation of therapy, 20 children treated with CGP56697 and one of the P/S-treated children returned with second malaria episodes (P < 0.0001). Genotyping suggested that the majority (19 of 23 [82.6%]) of these second episodes were due to new infections, supporting the World Health Organization recommendation that longer follow-up is not relevant for the assessment of drug efficacy. At the two-week follow-up, 28.9% of the P/S treated children but none of the CGP56697-treated children carried gametocytes (P < 0.0001). This study showed that CGP56697 is safe in African children with acute uncomplicated falciparum malaria, clears parasites more rapidly than P/S, and results in fewer gametocyte carriers. More frequent new infections within the third and fourth week following treatment with CGP56697 than treatment with P/S are likely to be due to the short prophylactic effect of CGP56697.

Polymorphism of the Pfmdr1 gene and chloroquine resistance in Plasmodium falciparum in The Gambia.

To assess the level of resistance to chloroquine (CQ) of Plasmodium falciparum in The Gambia in 1995-1996 we measured susceptibility in vivo by quantifying parasitaemia of children with mild malaria on days 4 and 8 after treatment. Pretreatment blood samples were used for susceptibility testing in vitro by the World Health Organization microtest and the prevalence of the tyrosine (tyr)86 allele of the Pfmdr1 gene was assessed by the polymerase chain reaction and restriction fragment length polymorphism analysis. Seven of 42 children (17%) treated with CQ remained parasitaemic on day 4 and required a change of antimalarial treatment. Susceptibility assays in vitro were performed on 50 P. falciparum isolates obtained from eligible children before treatment; 36 (72%) were resistant to CQ (> or = 1.6 mumol/L). The median minimum inhibitory concentration (MIC) of artemether was 3.38 nmol/L (range 0.42-13.51 nmol/L) and the median MIC of dihydroartemisinin was 0.88 nmol/L (range 0.22-14.04 nmol/L). Susceptibility in vitro to CQ and the Pfmdr1 genotype were determined for 31 fresh isolates. The allele was present in 12 of 22 isolates found to be resistant to CQ in vitro, but in none of the 9 isolates which were susceptible (Fisher's exact test, P = 0.005). All P. falciparum isolates with the tyr86 allele were CQ resistant in vitro, but since only half of all resistant isolates contained the allele, additional explanations for CQ resistance are required.

The safety of the combination artesunate and pyrimethamine-sulfadoxine given during pregnancy.

Malaria during pregnancy is associated with an increased risk of severe anaemia and low-birthweight babies. Effective intermittent therapy with pyrimethamine-sulfadoxine (PSD) decreases parasitaemia and severe anaemia and improves birthweight in areas where Plasmodium falciparum is sensitive to this drug. Increasing resistance to PSD is a concern and alternative antimalarial regimens during pregnancy are needed. Artesunate with PSD is a promising antimalarial combination but few data are available on the safety of artemisinins when taken during pregnancy. Outcome of pregnancy was evaluated for 287 women in The Gambia who were exposed in June 1999 to a single dose of the combination artesunate and PSD during a mass drug administration and 172 women who were not exposed. Women who received placebo (40) and those who did not participate in the mass drug administration (132) comprised the non-exposed group. There was no difference in the proportion of abortions, stillbirths, or infant deaths among those exposed or not exposed to the drugs. The mean weight of 18 infants born to mothers who had received artesunate and PSD during the third trimester was 3.10 kg compared to a mean weight of 2.62 kg of the 10 infants of untreated mothers (adjusted P value = 0.05). We found no evidence of a teratogenic or otherwise harmful effect of gestational exposure to artesunate and PSD. Treatment of a self-selected group of pregnant women with PSD and artesunate during pregnancy was associated with a greater birthweight, which may have resulted from clearance of malaria parasites. However, the influence of confounding factors cannot be excluded.

A randomized safety and tolerability trial of artesunate plus sulfadoxine--pyrimethamine versus sulfadoxine-pyrimethamine alone for the treatment of uncomplicated malaria in Gambian children.

Artemisinin derivatives, such as artesunate, have a short half-life and very rapid anti-malarial activity. Theoretically, using such agents in conjunction with well-established anti-malarial drugs such as sulfadoxine-pyrimethamine may reduce the rate of drug resistance. Such a combination has not previously been used in Africa. We have conducted a pilot safety trial of artesunate (4 mg/kg for 3 days) given with a single dose of sulfadoxine-pyrimethamine (25 mg/kg sulfadoxine) compared to sulfadoxine-pyrimethamine alone among 40 Gambian children with uncomplicated malaria. Both regimens were safe and well tolerated and there were no adverse experiences attributed to the combination. The addition of artesunate resulted in a higher proportion of afebrile children and children with a negative blood film on Day 2, and a reduction in the proportion of gametocyte carriers, when compared to sulfadoxine-pyrimethamine alone.

A study protocol for a randomised open-label clinical trial of artesunate-mefloquine versus chloroquine in patients with non-severe Plasmodium knowlesi malaria in Sabah, Malaysia (ACT KNOW trial).

INTRODUCTION: Malaria due to Plasmodium knowlesi is reported throughout South-East Asia, and is the commonest cause of it in Malaysia. P. knowlesi replicates every 24 h and can cause severe disease and death. Current 2010 WHO Malaria Treatment Guidelines have no recommendations for the optimal treatment of non-severe knowlesi malaria. Artemisinin-combination therapies (ACT) and chloroquine have each been successfully used to treat knowlesi malaria; however, the rapidity of parasite clearance has not been prospectively compared. Malaysia's national policy for malaria pre-elimination involves mandatory hospital admission for confirmed malaria cases with discharge only after two negative blood films; use of a more rapidly acting antimalarial agent would have health cost benefits. P. knowlesi is commonly microscopically misreported as P. malariae, P. falciparum or P. vivax, with a high proportion of the latter two species being chloroquine-resistant in Malaysia. A unified ACT-treatment protocol would provide effective blood stage malaria treatment for all Plasmodium species. METHODS AND ANALYSIS: ACT KNOW, the first randomised controlled trial ever performed in knowlesi malaria, is a two-arm open-label trial with enrolments over a 2-year period at three district sites in Sabah, powered to show a difference in proportion of patients negative for malaria by microscopy at 24 h between treatment arms (clinicaltrials.gov #NCT01708876). Enrolments started in December 2012, with completion expected by September 2014. A total sample size of 228 is required to give 90% power (α 0.05) to determine the primary end point using intention-to-treat analysis. Secondary end points include parasite clearance time, rates of recurrent infection/treatment failure to day 42, gametocyte carriage throughout follow-up and rates of anaemia at day 28, as determined by survival analysis. ETHICS AND DISSEMINATION: This study has been approved by relevant institutional ethics committees in Malaysia and Australia. Results will be disseminated to inform knowlesi malaria treatment policy in this region through peer-reviewed publications and academic presentations. TRIAL REGISTRATION NUMBER: NCT01708876.

Factors that are associated with the risk of acquiring Plasmodium knowlesi malaria in Sabah, Malaysia: a case-control study protocol.

INTRODUCTION: Plasmodium knowlesi has long been present in Malaysia, and is now an emerging cause of zoonotic human malaria. Cases have been confirmed throughout South-East Asia where the ranges of its natural macaque hosts and Anopheles leucosphyrus group vectors overlap. The majority of cases are from Eastern Malaysia, with increasing total public health notifications despite a concurrent reduction in Plasmodium falciparum and P. vivax malaria. The public health implications are concerning given P. knowlesi has the highest risk of severe and fatal disease of all Plasmodium spp in Malaysia. Current patterns of risk and disease vary based on vector type and competence, with individual exposure risks related to forest and forest-edge activities still poorly defined. Clustering of cases has not yet been systematically evaluated despite reports of peri-domestic transmission and known vector competence for human-to-human transmission. METHODS AND ANALYSIS: A population-based case-control study will be conducted over a 2-year period at two adjacent districts in north-west Sabah, Malaysia. Confirmed malaria cases presenting to the district hospital sites meeting relevant inclusion criteria will be requested to enrol. Three community controls matched to the same village as the case will be selected randomly. Study procedures will include blood sampling and administration of household and individual questionnaires to evaluate potential exposure risks associated with acquisition of P. knowlesi malaria. Secondary outcomes will include differences in exposure variables between P. knowlesi and other Plasmodium spp, risk of severe P. knowlesi malaria, and evaluation of P. knowlesi case clustering. Primary analysis will be per protocol, with adjusted ORs for exposure risks between cases and controls calculated using conditional multiple logistic regression models. ETHICS: This study has been approved by the human research ethics committees of Malaysia, the Menzies School of Health Research, Australia, and the London School of Hygiene and Tropical Medicine, UK.

Population pharmacokinetics of intramuscular artesunate in African children with severe malaria: implications for a practical dosing regimen.

Parenteral artesunate (ARS) is the drug of choice for the treatment of severe malaria. Pharmacokinetics data on intramuscular ARS are limited with respect to the main treatment group that carries the highest mortality, namely, critically ill children with severe malaria. A population pharmacokinetic study of ARS and dihydroartemisinin (DHA) was conducted from sparse sampling in 70 Tanzanian children of ages 6 months to 11 years. All the children had been admitted with severe falciparum malaria and were treated with intramuscular ARS (2.4 mg/kg at 0, 12, and 24 h). Venous plasma concentration-time profiles were characterized using nonlinear mixed-effects modeling (NONMEM). A one-compartment disposition model accurately described first-dose population pharmacokinetics of ARS and DHA. Body weight significantly affected clearance and apparent volume of distribution (P < 0.001), resulting in lower ARS and DHA exposure levels in smaller children. An adapted dosing regimen including a practical dosing table per weight band is proposed for young children based on the pharmacokinetic model.

Approaching the community about screening children for a multicentre malaria vaccine trial.

Community sensitisation, as a component of community engagement, plays an important role in strengthening the ethics of community-based trials in developing countries and is fundamental to trial success. However, few researchers have shared their community sensitisation strategies and experiences. We report on our perspective as researchers on the sensitisation activities undertaken for a phase II malaria vaccine trial in Kilifi District (Kenya) and Korogwe District (Tanzania), with the aim of informing and guiding the operational planning of future trials. We report wide variability in recruitment rates within both sites; a variability that occurred against a backdrop of similarity in overall approaches to sensitisation across the two sites but significant differences in community exposure to biomedical research. We present a range of potential factors contributing to these differences in recruitment rates, which we believe are worth considering in future community sensitisation plans. We conclude by arguing for carefully designed social science research around the implementation and impact of community sensitisation activities.

Molecular characteristics of Shigella spp. isolated from patients with diarrhoea in a new industrialized area of Thailand.

In this study, we used plasmid profile analysis, XbaI macrorestriction with pulsed-field gel electrophoresis (PFGE), and PCR of the ipaH gene, to study the molecular characteristics of 183 Shigella spp. isolated during May 2000 to April 2003 from rectal swabs of patients with watery and/or bloody diarrhoea in a new industrialized area of Thailand. Among the 183 isolates, 167 were S. sonnei and 16 were S. flexneri. For plasmid profile analysis, the 183 isolates revealed 16 different plasmid patterns, designated patterns A to P. The sizes of the plasmid bands were: 6, 5.5, 5, 4.5, 4, 3.25, 2.75, 2.5, 2, 1.75, 1.5 and/or 1.25 kb. The frequency of each plasmid band was 4.5 kb (165 isolates), 3.25 kb (161 isolates), 5.5 kb (129 isolates), 1.75 kb (121 isolates), 1.5 kb (35 isolates), 5 kb (21 isolates), 2 kb (16 isolates), 2.75 kb (12 isolates), 1.25 kb (9 isolates), and 6 kb (8 isolates). PFGE analysis revealed 45 different XbaI macrorestricted DNA banding patterns which could be grouped into 11 groups. All the isolates gave PCR amplicons of the ipaH gene. Plasmid profile analysis and PFGE are powerful tools for differentiation of the Shigella spp. This study provides important data on the molecular characteristics of Shigella isolates in Thailand, which could be useful as an epidemiological baseline for identifying relationships with strains that may emerge in the future.

Shigellosis remains an important problem in children less than 5 years of age in Thailand.

This is a review of existing data on the burden of shigellosis in Thailand to determine trends, vulnerable groups, predominant species and serotypes, and antimicrobial resistance patterns. Diarrhoea and dysentery morbidity and mortality data from 1991 to 1999 was collected from the routine surveillance system and demographic data from the government census. International and local literature published between 1988 and 2000 was systematically reviewed. Based on the routine surveillance system, the annual incidence of bacillary dysentery decreased from 1.3 to 0.2/10,000 persons per year. The remaining burden is highest in children <5 years of age at 2.7/10,000 persons per year. In comparison, a prospective study utilizing active surveillance found an incidence in children <5 years of age that was more than 100-fold higher at 640/10,000 persons per year. Despite the decrease in morbidity and mortality based on routinely collected data, shigellosis remains an important problem in children <5 years of age in Thailand.

The burden of cholera in the slums of Kolkata, India: data from a prospective, community based study.

AIMS: To conduct a prospective, community based study in an impoverished urban site in Kolkata (formerly Calcutta) in order to measure the burden of cholera, describe its epidemiology, and search for potential risk factors that could be addressed by public health strategies. METHODS: The study population was enumerated at the beginning and end of the study period. Surveillance through five field outposts and two referral hospitals for acute, watery, non-bloody diarrhoea was conducted from 1 May 2003 to 30 April 2004. Data and a stool sample for culture of Vibrio cholerae were collected from each patient. Treatment was provided in accordance with national guidelines. RESULTS: From 62 329 individuals under surveillance, 3284 diarrhoea episodes were detected, of which 3276 (99%) had a stool sample collected and 126 (4%) were culture confirmed cholera. Nineteen (15%) were children less than 2 years of age, 29 (23%) had severe dehydration, and 48 (38%) were hospitalised. Risk factors for cholera included a household member with cholera during the period of surveillance, young age, and lower educational level. CONCLUSIONS: There was a substantial burden of cholera in Kolkata with risk factors not easily amenable to intervention. Young children bear the brunt not only of diarrhoeal diseases in general, but of cholera as well. Mass vaccination could be a potentially useful tool to prevent and control seasonal cholera in this community.

Increased risk for malaria in chronically malnourished children under 5 years of age in rural Gambia.

Malaria and malnutrition cause high morbidity and mortality in rural sub-Saharan Africa. To explore the relationship between nutritional status and malaria, a cohort of Gambian children under 5 years of age was followed weekly during one malaria season. Anthropometric measurements were made at the beginning and at the end of the season. A total of 55/107 (51.4 per cent) children with baseline stunting, defined as having a height-for-age z-score below -2 standard deviations, subsequently experienced malaria episodes, compared to 145/380 (38.2 per cent) children who were not stunted (RR = 1.35; 95 per cent CI, 1.08-1.69; p value = 0.01). Neither wasting (weight-for-height z-score below -2 standard deviations) nor undernutrition (weight-for-age z-score below -2 standard deviations) influenced susceptibility to malaria. Adjustment for characteristics of age, sex, and ethnicity did not significantly change the risk ratios. Malaria had no effect on the nutritional status from the beginning to the end of the malaria season. Our findings suggest that chronically malnourished children may be at higher risk for developing malaria episodes.

Parasitaemia and gametocytaemia after treatment with chloroquine, pyrimethamine/sulfadoxine, and pyrimethamine/sulfadoxine combined with artesunate in young Gambians with uncomplicated malaria.

As part of a study to assess the infectivity of gametocytes after treatment with four antimalarial regimens, the efficacy of each treatment was also determined. From September to December 1998, 598 children with uncomplicated malaria were treated; 135 received chloroquine (CQ) alone, 276 received pyrimethamine/sulfadoxine (Fansidar, PSD) alone, 113 received PSD with a single dose of artesunate (PSD + 1ART) and 74 received PSD combined with three doses of artesunate (PSD + 3ART). On day 28 19/63 (30.2%; 95% C.I. 19.2% to 43.1%) of children treated with CQ alone, 5/134 (3.7%; 95% C.I. 1.2% to 8.5%) treated with PSD alone, 1/71 (1.4%, 95% C.I. 0.0% to 7.9%) treated with PSD + 1ART and 0/45 (0.0%; 95% C.I. 0.0% to 7.9%) treated with PSD + 3ART were parasitaemic. The proportion of children with gametocytes on day 7 after treatment with CQ alone was 16/89 (18.0%; 95% C.I. 10.6% to 27.6%), 98/174 (56.3%; 95% C.I. 48.6% to 63.8%) after treatment with PSD alone, 8/70 (11.4%; 95% C.I. 5.1% to 21.3%) after treatment with PSD + 1ART and 4/46 (8.7%; 95% C.I., 2.4% to 20.8%) after treatment with PSD + 3ART. CQ thus has a lower efficacy than PSD or either of the PSD and artesunate combinations. Use of PSD alone as an alternative first line treatment results in a very high post-treatment gametocyte prevalence that is likely to enhance transmission. There would be greater and more sustainable benefits from using PSD and artesunate combinations.

Inoculum effect leads to overestimation of in vitro resistance for artemisinin derivatives and standard antimalarials: a Gambian field study.

Artemisinin (QHS) and its derivatives are new antimalarials which are effective against Plasmodium falciparum parasites resistant to chloroquine (CQ). As these drugs are introduced it is imperative that resistance is monitored. In this paper we demonstrate that the inoculum size used in in vitro testing influences the measured in vitro susceptibility to QHS and its derivative dihydroartemisinin (DHA) and to mefloquine (MEF) and CQ over the range of parasitaemias routinely used in testing with the WHO in vitro microtest. An increase in parasitaemia and/or haematocrit was accompanied by a decrease in the measured sensitivity of 2 laboratory lines. In the context of a field study testing in vitro susceptibility of parasite isolates from patients with uncomplicated malaria in Fajara, The Gambia we demonstrate that failure to control for inoculum size significantly overestimates the level of resistance to QHS and DHA as well as MEF, halofantrine (HAL) and quinine (QUIN). When controlling for the inoculum effect, cross-resistance was observed between QHS, MEF and HAL suggesting the presence of a multidrug resistance-like mechanism. These studies underline the importance of inoculum size in in vitro susceptibility testing.