Krishanthi Subramaniam

The absence of serum IgM enhances the susceptibility of mice to pulmonary challenge with Cryptococcus neoformans.

The importance of T cell-mediated immunity for resistance to the disease (cryptococcal disease) caused by Cryptococcus neoformans is incontrovertible, but whether Ab immunity also contributes to resistance remains uncertain. To investigate the role of IgM in resistance to C. neoformans, we compared the survival, fungal burden, lung and brain inflammatory responses, and lung phagocytic response of sIgM−/− mice, which lack secreted IgM, to that of IgM sufficient C57BL6x129Sv (heretofore, control) mice at different times after intranasal infection with C. neoformans (24067). sIgM−/− mice had higher mortality and higher blood and brain CFUs 28 d postinfection, but lung CFUs were comparable. Lungs of control mice manifested exuberant histiocytic inflammation with visible C. neoformans, findings that were not observed in sIgM−/− mice, whereas in brain sections, sIgM−/− mice had marked inflammation with visible C. neoformans that was not observed in control mice. Cytokine responses were significant for higher levels of lung IL-1β and IL-12 24 h postinfection in control mice and higher levels of lung and brain IL-17 28 d postinfection in sIgM−/− mice. Alveolar macrophage phagocytosis was significantly higher for control than for sIgM−/− mice 24 h postinfection; however, phagocytic indices of sIgM−/− mice increased after reconstitution of sIgM−/− mice with polyclonal IgM. These data establish a previously unrecognized role for IgM in resistance to intranasal infection with C. neoformans in mice and suggest that the mechanism by which it mediates a host benefit is by augmenting Th1 polarization, macrophage recruitment and phagocytosis of C. neoformans.

IgM+ Memory B Cell Expression Predicts HIV-Associated Cryptococcosis Status

BACKGROUND The role of B cells in resistance to Cryptococcus neoformans disease (i.e., cryptococcosis) is unknown. Given evidence that IgM(+) memory B cells are required for immunity to other encapsulated pathogens, we hypothesized that these cells might contribute to resistance to cryptococcosis. METHODS We compared levels of IgM expression on memory B cells in 29 HIV-infected individuals who had a history of cryptococcosis (the HIV+CN+ group) with levels in 30 human immunodeficiency virus (HIV)-infected subjects who had no history of cryptococcosis (the HIV+CN- group) and 20 HIV-uninfected subjects who had no history of cryptococcosis (the HIV- group) (cohort 1). We also determined levels of IgM expression on memory B cells in banked samples obtained before cryptococcosis onset from 31 participants in the Multicenter AIDS Cohort Study, of whom 8 had HIV infection and subsequently developed cryptococcosis (the HIV+CN+ group), 8 had HIV infection and did not develop cryptococcosis (the HIV+CN- group), and 15 did not have HIV infection and did not develop cryptococcosis (the HIV- group) (cohort 2). RESULTS In cohort 1, the percentage of memory B cells that expressed IgM was lower among HIV+CN+ subjects, compared with HIV+CN- subjects (P < .01) and HIV- subjects (P < .05); expression of IgM on 50% of memory B cells was a significant predictor of C. neoformans disease status (odds ratio, 5.5; P = .03). In cohort 2, the percentage of memory B cells that expressed IgM was lower in HIV+CN+ subjects than in HIV+CN- subjects (P = .02) and HIV- subjects (P < .01); an IgM(+) memory B cell percentage of 38.5% was a significant predictor of future development of cryptococcosis (odds ratio, 14; P = .02). CONCLUSIONS These findings suggest that HIV-infected persons in whom the percentage of memory B cells that express IgM is decreased might be at greater risk for the development of cryptococcosis.

Protective and nonprotective human immunoglobulin M monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan manifest different specificities and gene use profiles

ABSTRACT The features of protective murine antibodies to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been rigorously investigated; however, the characteristics of protective human antibodies to GXM have not been defined. We produced monoclonal antibodies (MAbs) from XenoMouse mice (transgenic mice that express human immunoglobulin M [IgM], IgG2, and κ) which were immunized with a C. neoformans serotype D strain 24067 GXM-diphtheria toxoid conjugate. This study reports the specificity and efficacy of three human IgM MAbs, G14, G15, and G19, generated from these mice. Each MAb was specific for GXM, but G14 and G19 had different specificity based on their binding to serotype A strain H99 and SB4 GXMs, to which G15 did not bind. Nucleic acid sequence analysis revealed that G15 uses VH3-64 in the germ line configuration. G14 and G19 use VH6-1, which has somatic mutations. All of the MAbs use Vκ DPK22/A27. Studies of MAb efficacy in BALB/c mice showed that administration of 0.1 mg, but not 1 or 0.01 mg, of G15 prolonged survival against lethal C. neoformans strain 24067 challenge, whereas G14 and G19 were not protective at any dose. This panel of MAbs illustrates that serotype D GXM has epitopes that elicit human antibodies that can be either protective or nonprotective. Our findings suggest that VH gene use may influence GXM specificity and efficacy, and they provide insights into the possible contribution that VH gene use may have in resistance and susceptibility to cryptococcosis.

Design of a study to determine the impact of insecticide resistance on malaria vector control: a multi-country investigation.

BackgroundProgress in reducing the malaria disease burden through the substantial scale up of insecticide-based vector control in recent years could be reversed by the widespread emergence of insecticide resistance. The impact of insecticide resistance on the protective effectiveness of insecticide-treated nets (ITN) and indoor residual spraying (IRS) is not known. A multi-country study was undertaken in Sudan, Kenya, India, Cameroon and Benin to quantify the potential loss of epidemiological effectiveness of ITNs and IRS due to decreased susceptibility of malaria vectors to insecticides. The design of the study is described in this paper.MethodsMalaria disease incidence rates by active case detection in cohorts of children, and indicators of insecticide resistance in local vectors were monitored in each of approximately 300 separate locations (clusters) with high coverage of malaria vector control over multiple malaria seasons. Phenotypic and genotypic resistance was assessed annually. In two countries, Sudan and India, clusters were randomly assigned to receive universal coverage of ITNs only, or universal coverage of ITNs combined with high coverage of IRS. Association between malaria incidence and insecticide resistance, and protective effectiveness of vector control methods and insecticide resistance were estimated, respectively.ResultsCohorts have been set up in all five countries, and phenotypic resistance data have been collected in all clusters. In Sudan, Kenya, Cameroon and Benin data collection is due to be completed in 2015. In India data collection will be completed in 2016.DiscussionThe paper discusses challenges faced in the design and execution of the study, the analysis plan, the strengths and weaknesses, and the possible alternatives to the chosen study design.

Cutaneous Leishmaniasis and Conflict in Syria.

To the Editor: War, infection, and disease have always made intimate bedfellows, with disease recrudescence characterizing most conflict zones (1). Recently, increasing violence from civil war and terrorist activity in the Middle East has caused the largest human displacement in decades. A neglected consequence of this tragedy has been the reemergence of a cutaneous leishmaniasis epidemic. Old World cutaneous leishmaniasis is one of the most prevalent insectborne diseases within the World Health Organization’s Eastern Mediterranean Region (2). Zoonotic cutaneous leishmaniasis is caused by the protozoan parasite Leishmania major, which is transmitted through the infectious bite of the female Phlebotomus papatasi sand fly; the animal reservoirs are the rodent genera Rhombomys, Psammomys, and Meriones. Anthroponotic cutaneous leishmaniasis is caused by L. tropica and transmitted between humans by the Ph. sergenti sand fly. Until 1960, cutaneous leishmaniasis prevalence in Syria was restricted to 2 areas to which it is endemic (Aleppo and Damascus); preconflict (c. 2010) incidence was 23,000 cases/year (3). However, in early 2013, an alarming increase to 41,000 cutaneous leishmaniasis cases was reported (3,4). The regions most affected are under Islamic State control; 6,500 cases occurred in Ar-Raqqah, Diyar Al-Zour, and Hasakah. Because these places are not historical hotspots of cutaneous leishmaniasis, this change might be attributed to the massive human displacement within Syria and the ecologic disruption of sand fly (Ph. papatasi) habitats. According to the United Nations High Commissioner for Refugees, >4.2 million Syrians have been displaced into neighboring countries; Turkey, Lebanon, and Jordan have accepted most of these refugees. As a result, cutaneous leishmaniasis has begun to emerge in areas where displaced Syrians and disease reservoirs coexist (5). According to the Lebanese Ministry of Health, during 2000–2012, only 6 cutaneous leishmaniasis cases were reported in Lebanon. However in 2013 alone, 1,033 new cases were reported, of which 96.6% occurred among the displaced Syrian refugee populations (5). Similarly in Turkey, nonendemic parasite strains L. major and L. donovani were introduced by incoming refugees (6). Many of the temporary refugee settlements are predisposed to increased risk because of malnutrition, poor housing, absence of clean water, and inadequate sanitation. The combination of favorable climate, abundant sand fly populations, displaced refugees, and deficient medical facilities and services has created an environment conducive to cutaneous leishmaniasis reemergence. For example, refugee settlements in Nizip in southern Turkey have reported several hundred cases (7). Using current datasets published in English and Arabic, we mapped cutaneous leishmaniasis prevalence within Syria and its neighboring countries (Figure). Our results demonstrate that cutaneous leishmaniasis prevalence coincides with the presence of refugee camps (Figure, panel A), which is plausible given the strong association between disease outbreaks and refugee settlements (8). The deterioration of Syrian health systems, including the cessation of countrywide vector control programs, has created an ideal environment for disease outbreaks (9). Likewise, the sand fly vectors are widely distributed throughout the Middle East; expansive Ph. papatasi and Ph. sergenti sand fly populations exist in Syria and Iraq (4). The presence of these vectors in regions of instability can create new cutaneous leishmaniasis foci, which might have debilitating, and often stigmatizing, consequences for residents and deployed military personnel (10). In addition, the distribution of Leishmania spp. overlaps with sand fly habitats (Figure, panel B) and disease reservoirs (W. Al-Salem, unpub. data). Consequently, the movement of large refugee populations into regions that are ill-equipped to manage imported cutaneous leishmaniasis has resulted in outbreaks in Turkey and Lebanon (5,6). Figure Cutaneous leishmaniasis prevalence within Syria and neighboring countries of the World Health Organization’s Eastern Mediterranean Region, 2013. A) Prevalence among refugee camps. Case data were taken from http://datadryad.org/resource/doi:10.5061/dryad.05f5h ... Our findings emphasize the importance of contemporaneous disease tracking to identify human populations at highest disease risk. To ameliorate the current cutaneous leishmaniasis crisis, particularly during the winter when cases start to appear, accurate disease monitoring and strategic training of persons based within refugee camps (medical staff, aid workers, volunteers, and military personnel) needs to be prioritized. Moreover, clinicians and other medical personnel residing in refugee-hosting countries must be suitably trained to diagnose cutaneous leishmaniasis because other local diseases (e.g., sarcoidosis and cutaneous tuberculosis) can have similar manifestations. Along with vector and rodent control, new cutaneous leishmaniasis outbreaks should be managed by prompt diagnosis and treatment, which are even more pertinent given that L. tropica–associated cutaneous leishmaniasis typically is resistant to several treatment regimens. In summary, the coexistence of sand fly populations and Leishmania spp. within refugee camps, together with the considerable influx of persons who already have cutaneous leishmaniasis, create a dangerous cocktail that can lead to an outbreak unprecedented in modern times.

Insecticide-Treated Nets and Protection against Insecticide-Resistant Malaria Vectors in Western Kenya

Insecticide resistance might reduce the efficacy of malaria vector control. In 2013 and 2014, malaria vectors from 50 villages, of varying pyrethroid resistance, in western Kenya were assayed for resistance to deltamethrin. Long-lasting insecticide-treated nets (LLIN) were distributed to households at universal coverage. Children were recruited into 2 cohorts, cleared of malaria-causing parasites, and tested every 2 weeks for reinfection. Infection incidence rates for the 2 cohorts were 2.2 (95% CI 1.9–2.5) infections/person-year and 2.8 (95% CI 2.5–3.0) infections/person-year. LLIN users had lower infection rates than non-LLIN users in both low-resistance (rate ratio 0.61, 95% CI 0.42–0.88) and high-resistance (rate ratio 0.55, 95% CI 0.35–0.87) villages (p = 0.63). The association between insecticide resistance and infection incidence was not significant (p = 0.99). Although the incidence of infection was high among net users, LLINs provided significant protection (p = 0.01) against infection with malaria parasite regardless of vector insecticide resistance.

HIV Malaria Co-Infection Is Associated with Atypical Memory B Cell Expansion and a Reduced Antibody Response to a Broad Array of Plasmodium falciparum Antigens in Rwandan Adults

HIV infected individuals in malaria endemic areas experience more frequent and severe malaria episodes compared to non HIV infected. This clinical observation has been linked to a deficiency in antibody responses to Plasmodium falciparum antigens; however, prior studies have only focused on the antibody response to <0.5% of P. falciparum proteins. To obtain a broader and less-biased view of the effect of HIV on antibody responses to malaria we compared antibody profiles of HIV positive (HIV+) and negative (HIV-) Rwandan adults with symptomatic malaria using a microarray containing 824 P. falciparum proteins. We also investigated the cellular basis of the antibody response in the two groups by analyzing B and T cell subsets by flow cytometry. Although HIV malaria co-infected individuals generated antibodies to a large number of P. falciparum antigens, including potential vaccine candidates, the breadth and magnitude of their response was reduced compared to HIV- individuals. HIV malaria co-infection was also associated with a higher percentage of atypical memory B cells (MBC) (CD19+CD10-CD21-CD27-) compared to malaria infection alone. Among HIV+ individuals the CD4+ T cell count and HIV viral load only partially explained variability in the breadth of P. falciparum-specific antibody responses. Taken together, these data indicate that HIV malaria co-infection is associated with an expansion of atypical MBCs and a diminished antibody response to a diverse array of P. falciparum antigens, thus offering mechanistic insight into the higher risk of malaria in HIV+ individuals.

Presence of the knockdown resistance mutation, Vgsc-1014F in Anopheles gambiae and An. arabiensis in western Kenya

IntroductionThe voltage gated sodium channel mutation Vgsc-1014S (kdr-east) was first reported in Kenya in 2000 and has since been observed to occur at high frequencies in the local Anopheles gambiae s.s. population. The mutation Vgsc-1014F has never been reported from An. gambiae Complex complex mosquitoes in Kenya.FindingsMolecularly confirmed An. gambiae s.s. (hereafter An. gambiae) and An. arabiensis collected from 4 different parts of western Kenya were genotyped for kdr from 2011 to 2013. Vgsc-1014F was observed to have emerged, apparently, simultaneously in both An. gambiae and An. arabiensis in 2012. A portion of the samples were submitted for sequencing in order to confirm the Vgsc-1014F genotyping results. The resulting sequence data were deposited in GenBank (Accession numbers: KR867642-KR867651, KT758295-KT758303). A single Vgsc-1014F haplotype was observed suggesting, a common origin in both species.ConclusionThis is the first report of Vgsc-1014F in Kenya. Based on our samples, the mutation is present in low frequencies in both An. gambiae and An. arabiensis. It is important that we start monitoring relative frequencies of the two kdr genes so that we can determine their relative importance in an area of high insecticide treated net ownership.

Impact of insecticide resistance in Anopheles arabiensis on malaria incidence and prevalence in Sudan and the costs of mitigation.

Significance Emerging insecticide resistance in malaria vectors could presage a catastrophic rebound in malaria morbidity and mortality. In areas of moderate levels of resistance to pyrethroids, long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) with a carbamate insecticide were significantly more effective than IRS with pyrethroid insecticide. The impact on the effectiveness of LLINs could not be quantified. The incremental cost of using a carbamate insecticide to which vectors are susceptible was US

An α-Gal-containing neoglycoprotein-based vaccine partially protects against murine cutaneous leishmaniasis caused by Leishmania major

0.65 per person protected per year, which is considered acceptable by international standards. While the WHO recommends that different interventions, where possible, should use different insecticide classes, these data alone should not be used as the basis for a policy change in vector control interventions. Insecticide-based interventions have contributed to ∼78% of the reduction in the malaria burden in sub-Saharan Africa since 2000. Insecticide resistance in malaria vectors could presage a catastrophic rebound in disease incidence and mortality. A major impediment to the implementation of insecticide resistance management strategies is that evidence of the impact of resistance on malaria disease burden is limited. A cluster randomized trial was conducted in Sudan with pyrethroid-resistant and carbamate-susceptible malaria vectors. Clusters were randomly allocated to receive either long-lasting insecticidal nets (LLINs) alone or LLINs in combination with indoor residual spraying (IRS) with a pyrethroid (deltamethrin) insecticide in the first year and a carbamate (bendiocarb) insecticide in the two subsequent years. Malaria incidence was monitored for 3 y through active case detection in cohorts of children aged 1 to <10 y. When deltamethrin was used for IRS, incidence rates in the LLIN + IRS arm and the LLIN-only arm were similar, with the IRS providing no additional protection [incidence rate ratio (IRR) = 1.0 (95% confidence interval [CI]: 0.36–3.0; P = 0.96)]. When bendiocarb was used for IRS, there was some evidence of additional protection [interaction IRR = 0.55 (95% CI: 0.40–0.76; P < 0.001)]. In conclusion, pyrethroid resistance may have had an impact on pyrethroid-based IRS. The study was not designed to assess whether resistance had an impact on LLINs. These data alone should not be used as the basis for any policy change in vector control interventions.